Question Bank with Answer

Enzyme & Protein Engg. (EBT-021)

Unit I

1. Q. What do you mean by stability of enzyme?Answer: Enzymes are often fragile molecules that need to be protected during purification and characterization. Enzyme denaturation refers the loss of enzyme structure due to unfolding. Maintaining biological activity is often important and enzyme denaturation should be avoided in those situations. Elevated temperatures, extremes in pH, and changes in chemical or physical environment can all lead to enzyme denaturation . In general, things that destabilize H-bonding and other forces that contribute to secondary and tertiary enzyme structure will promote enzyme denaturation. Different enzymes exhibit different degrees of sensitivity to denaturing agents and some enzymes can be re-folded to their correct conformations following denaturation.

2. Q. What is the Factors Affecting Enzyme/Protein Stability?

Answer: Factors Affecting Enzyme/Protein Stability

3. Q. What is the optimum temperature for Enzyme/Protein Stability?

The optimal conditions for maintaining the stability of each individual enzyme need to be determined empirically. In general, though, enzyme solutions should be kept cold (< 4oC) except during assays and other procedures requiring specific temperatures. Many enzymes are especially labile and need to be stored at -20oC or -80oC. However, repeated freezing and thawing of enzyme solutions is often deleterious. Adding 50% glycerol to storage buffers will lower the freezing point and allow storage at -20oC. Solutions for working with enzymes will often contain heavy-metal chelators and/or antioxidants as protectants for enzymes. In addition, proteases may be released during cell disruption and it may therefore be necessary to include protease inhibitors.

4. Q. What is General Factors Affecting Enzyme/Protein stability? Explain

General Factors Affecting Enzyme/Protein stability

The native three-dimensional conformation of a protein is maintained by a rang of noncovalent interactions (electrostatic forces, hydrogen bonds, hydrophobic forces) and covalent interactions (disulfide bonds), in addition to the peptide bonds between individual amino acids.

Electrostatic forces: these include the interactions between two ionic groups of opposite charge, for example the ammonium group of Lys and the carboxyl group of Asp, often referred to as an ion pair or salt bridge. In addition, the noncovalent associations between electrically neutral molecules, collectively referred to as van der Waals forces, arise from electrostatic interactions between permanent and/or induced dipoles, such as the carbonyl group in peptide bonds.

Hydrogen bonds: these are predominantly electrostatic interactions between a weakly acidic donor group and an acceptor atom that bears a lone pair of electrons, which thus has a partial negative charge that attracts the hydrogen atom. In biological systems the donor group is an oxygen or nitrogen atom that has a covalently attached hydrogen atom, and the acceptor is eitheroxygen or nitrogen (Fig. 9). Hydrogen bonds are normally in the range 0.27–0.31 nm and are highly directional, i.e. the donor, hydrogen and acceptor atoms are colinear. Hydrogen bonds are stronger than van der Waals forces but much weaker than covalent bonds. Hydrogen bonds not only play an important role in protein structure, but also in the structure of other biologicalmacromolecules such as the DNAdouble helix and lipid bilayers . In addition, hydrogen bonds are critical to both the properties of water and to its role as a biochemical solvent.

Hydrophobic forces: The hydrophobic effect is the name given to those forces that cause non-polar molecules to minimize their contact with water. This is clearly seen with amphipathic molecules such as lipids and detergents which form micelles in aqueous solution . Proteins, too, find a conformation in which their non-polar side chains are largely out of contact with the aqueous solvent, and thus hydrophobic forces are an important determinant of protein structure, folding and stability. In proteins, the effects of

hydrophobic forces are often termed hydrophobic bonding, to indicate the specific nature of protein folding under the influence of the hydrophobic effect.

Disulfide bonds: These covalent bonds form between Cys residues that are close together in the final conformation of the protein and function to stabilize its three-dimensional structure. Disulfide bonds are really only formed in the oxidizing environment of the endoplasmic reticulum, and thus are found primarily in extracellular and secreted proteins.

5. Q. What do you mean by hydrophobic effect?Answer: The hydrophobic effect is the name given to those forces that cause non-polar molecules to minimize their contact with water. This is clearly seen with amphipathic molecules such as lipids and detergents which form micelles in aqueous solution . Proteins, too, find a conformation in which their non-polar side chains are largely out of contact with the aqueous solvent, and thus hydrophobic forces are an important determinant of protein structure, folding and stability. In proteins, the effects of hydrophobic forces are often termed hydrophobic bonding, to indicate the specific nature of protein folding under the influence of the hydrophobic effect.

6. Q. How Enzyme stabilization by selection?Answer: Enzyme stabilization by selectionBiocatalysts are inherently labile; therefore their operational stability is very importance for any bioprocess. An industrial disadvantage of the most commercially used biocatalysts- enzymes and enzyme complexes - is their relatively low stability.

Selection of industrial enzymes from any sources is depends on the following factors: a. specificity b. pH c. thermostability d. activation or inhibition e. availability and cost

Therefore, microorganisms which require extreme physicochemical conditions for their growth and proliferation - and enzymes derived from them - bear a potential for overcoming this situation.

The particular, enzymes derived from thermophilic microorganisms show an increased thermal stability compared to those of mesophilic microorganisms. They are called thermostable if they have a maximum reaction temperature above that of the optimum growth temperature for the microorganism. This definition is invalid in view of the fact that enzymes from microorganisms growing at temperatures above 90 OC are "thermostable" regardless of whether their optimum activity temperature is below or above the optimum growth temperature. It becomes obvious that thermostability of an enzyme is a relative term.

Stability of the enzyme is a function of the stabilizing forces which include hydrogen bonding, hydrophobic bonding, ionic interactions, metal binding, and/or disulfide linkages . Such stabilizing effects contribute to the long-term stability of an enzyme. Thermostability is also connected with a higher resistance to most chemical denaturants. A suitable method for characterizing the enzyme stability is the scanning calorimetry.

Thermophilic microorganisms are a source of more stable enzymes. Mixed populations of bacteria which seemed to occur in the ocean near the Galapagos Island in a depth of 2500 metres were generally an optimum growth temperature is 250OC at a pressure of 250 atm .There is reliable verification of the existence of microorganisms at temperatures approaching the boiling point of water given in the (Table 1 )

Table 1. Optimum growth temperature ( Topt ) of thermophilic bacteria

Species Topt ( OC)

Bacillus acidocaldarius 60 - 65Bacillus stearothermophilus 55 - 70Caldarobacterium hydrogenophilum 74 - 76Clostridium thermohydrosulfuricum 67 - 70Methanobacterium thermolithotrophicum 65 - 70Pyrococcus furiosus 100Pyrodictium occultum 105Sulfolobus acidocaldarius 70 - 75Thermoproteus tenax 88Thermotoga maritime 80Thermus aquaticus 70

These bacteria occur in natural and/or artificial habitats. As an example the original isolations of Thermus aquaticus were from hot spring algal mats. Incubation in aerobic liquid medium at 70 up to 75 OC led to the formation of visible turbidity, often with clumps.

Brock and Freeze isolated one strain of Thermus aquaticus from a hot water tap in Indiana. Brock and Yoder found another strain of Thermus aquaticus in a creek receiving thermal pollution. Ramely and Hixson as well as Brock and Boylen and Heinritz et al. obtained strains of Thermus aquaticus in a creek receiving thermal pollution and domestic hot water reservoirs, respectively. It thus seems likely that Thermus is capable of growing in manmade habitats of high temperature.

Table 2 shows the specific growth rate and the specific yield coefficient of selected thermophilic bacteria utilizing glucose as the carbon and energy sources at temperatures up to 80OC.

Table 2. Specific growth rate (μ) specific yield coefficient ( Y x/ s), and optimum growth temperature (& topt ) of selected thermophilic microorganisms utilizing glucose.

From the table 2 we find that the specific growth rate is in the range of the values of mesophilic bacteria. On the other hand, the specific yield coefficient is obviously lower compared to mesophiles.

The specific growth rate and/or the specific yield coefficient of thermophilic bacteria could be improved by- continuous instead of batch cultivation preventing inhibition of bacterial growth and proliferation due to carbon substrate and/or metabolites- increasing system pressure in fermenters.

7. Q. Advantage of More stable enzymes from thermophilic microorganisms.

Answer: Advantage of More stable enzymes from thermophilic microorganisms. Thermostable enzymes have several advantages over their counterparts from mesophilic microorganisms- higher thermal stability and resistance to most of the chemical denatu-- higher storage stability, - increased reaction rate and comparable catalytic activity,- lower viscosity of reaction mixture and improved mass-transfer,- lower danger of contamination in microbial enzyme production as well as

Fig 1: Thermal stability of a thermostable β-galactosidase of Bacillus stearothermophilus TP32 compared to the mesophilic β-galactosidase of Escherichia coli

Fig 2: Resistance of the thermostable (β-galactosidase of Bacillus stearothermophilus TP 32 and the mesophilic β–galactosidase of Escherichia coli to ethanol and propan-2-ol under incubationof the enzyme for 60 min at 50OC

8. Q. What is the application of thermostable enzymesApplication of thermostable enzymes

Because of these facts thermostable carbohydratases, proteases, and oxidoreductases were introduced recently into starch processing, hydrolysis of cellulose and lactose, brewing, baking, food processing, waste water treatment, biosensors and/or other applications. Thermostable and highly specific enzymes, e.g. DNA polymerases and RNA polymerases open up new dimensions in molecular biology and genetic engineering. As an example the DNA polymerase of Thermus aquaticus made possible the polymerase chain reaction ( PCR ) technology.

9. Q. How Enzyme stabilization by genetic engineering?Enzyme stabilization by genetic engineering Genetic engineering and protein engineering are modern techniques already in use for the commercial production of biocatalysts of improved stability, not only to high temperatures, but also to extremes of pH, oxidizing agents and organic solvents. Cloning and expression in suitable hosts is being used routinely by major enzyme production companies to produce improved biocatalysts..

Protein engineering is also being used to obtain improved biocatalysts, the case of alkaline protease being a paradigm. Already in the market, thermostable proteases capable to withstand harsh washing conditions (high pH, high concentration of strong oxidants) are products of protein engineering produced by point amino acid substitutions in the most labile region of the molecules

In the production of syrups from cornstarch, thermostability of a-amylase is severely reduced below pH 6, which poses the inconvenience of pH adjustment before and after starch liquefaction. A thermostable a-

amylase from Bacillus licheniformis, active at low pH and low Ca++concentration has been recently patented. A thermostable glucose isomerase is a major challenge in the production of high-fructose corn syrup. Equilibrium is favored at high temperature, so that at 110 °C 55 % HFCS could be produced at the enzyme reactor stage, without the cumbersome process of sugar fractionation now used. It was shown that specific substitution of a surface arginine residue for lysine, obtained by site-directed mutagenesis, produced a substantial thermal stabilization in the glucose isomerase from Actinoplanes missouriensis.Protein engineering is a powerful tool for the design of robust biocatalysts and probably most future biocatalysts will be produced by engineered organisms.Genetic gngineering follows the convenience of cloning termophilic genes into more suitable mesophilic hosts. Those systems will be highly productive and the enzymes produced will retain its original thermostability. In fact, in a number of cases thermophilic genes have been cloned and expressed in mesophilic hosts, producing enzymes highly active and stable at high temperatures. Some examples are in Table below and other bacterial hosts pose some problems in expressing genes from archibacteria, because of misreading of intervened genes. E.coli and other bacterial hosts pose some problems in expressing genes from archibacteria, because of misreading of intervened genes. This is not the case with eubacterial genes, being therefore better candidates for cloning into bacterial hostsFollowing Industrial thermostable enzymes, commercial enzymes from thermophiles and termophilic genes cloned in mesophilic hosts given in the table below.

10. Q. What are the factors on which selection of industrial enzymes from any sources is depends?

Answer: Selection of industrial enzymes from any sources is depends on the following factors: a. specificity b. pH c. thermostability d. activation or inhibition e. availability and costQ. Explain the significance of sigmoidal behavior of enzyme?Answer:

11. Q. Explain one of the common examples of allostery.

12. Q . Write short notes on K-series and V-series allosteric enzyme.

13. Q. What do you mean by Enzyme & Protein engineering?

Answer: Protein/enzyme engineering refers to the effort to design new protein molecules of a desired 3-D structure and function. It is a reverse procedure of protein structure prediction and the solution of the problem therefore highly relies on the extent of our understanding on the principle of protein foldingTherefore targets for enzyme/ protein engineering are

I. enhancement of enzyme activity,II. improved stability of the protein,

III. altered pH optima or temperature tolerance and IV. modified specificity

14. Q. Describe the characteristic feature of enzyme engineering.Answer: As we know that large number of proteins/enzymes found in nature, which fold into a variety of different structures and carry out a huge diversity of functions. Protein engineering attempts to design protein/enzyme structures, including those having particular functions. Therefore targets for enzyme/ protein engineering are

· enhancement of enzyme activity,· improved stability of the protein,· altered pH optima or temperature tolerance and · modified specificity

Researchers can improve the knowledge we have about the forces and effects that specify

the properties of the folded states of a protein. In addition, control over the design of

particular folded state structures will likely lead to new synthetic proteins having the

efficiency and specificity of biological proteins.

Designing a new protein/enzyme molecule has applications include therapeutics, sensors,

catalysts, and materials. The successful design of proteins/enzymes is possible even without

a complete quantitative understanding of all the forces involved in specifying their

structures.

Designing of proteins/enzymes is nontrivial, however, because of both their complexity and

the delicacy of the interactions that specify the folded state. Proteins are macrobiomolecule

and having many structural variables specify the folded state, including sequence, backbone

topology, and side-chain conformations.

There are two main motivations for designing of proteins/enzymes:

The first is based upon the assumption that a complete understanding of any

natural system depend upon our ability to design a similar artificial system from first

principles.

The second motivation is for de novo protein design is one of the practical

approaches. Therefore our understanding of natural proteins for their folding

pathways, thermodynamic stabilities and catalytic properties is enhanced by our

ability to design novel proteins with predetermined structure and properties. The

ability to design proteins/enzymes de novo has the potential to bring a revolution in

the field of science and technology ranging from industrial catalyst to biomedical

engineering.

Protein/enzyme design also refers to the effort to design new protein molecules of a desired

3-D structure and function. It is a reverse procedure of protein structure prediction and the

solution of the problem therefore highly relies on the extent of our understanding on the

principle of protein folding.

How ?

Fig: Protein design is a reverse procedure of protein structure prediction.

Finding out an amino acid sequence that will adopt a unique and stable three dimensional

structure is the main goal to design a novel protein. The fundamental hurdle to design a

novel protein is the conformational entropy of the linear polymer chain which must be

overcome. The conformational entropy represents a substantial amount of unfavorable free

energy. For a design to succeed the favorable free energy associated with these designed

interactions must outweigh the entropic cost of fixing the chain into a unique structure.

15. Q. Why protein engineering important? Explain.

Answer: As we know that large number of proteins found in nature, which fold into a

variety of different structures and carry out a huge diversity of functions. Protein

engineering attempts to design protein structures, including those having particular

functions, researchers can improve the knowledge we have about the forces and effects that

specify the properties of the folded states of a protein. In addition, control over the design of

particular folded state structures will likely lead to new synthetic proteins having the

efficiency and specificity of biological proteins. Such applications include therapeutics,

sensors, catalysts, and materials. The successful design of proteins is possible even without

a complete quantitative understanding of all the forces involved in specifying their

structures.

Designing of proteins is nontrivial, however, because of both their complexity and the

subtlety of the interactions that specify the folded state. Proteins are macrobiomolecule and

having many structural variables specify the folded state, including sequence, backbone

topology, and side-chain conformations.

There are two main motivations for doing protein engineering. The first is based upon

the assumption that a complete understanding of any natural system depend upon our ability

to design a similar artificial system from first principles.

The second motivation is for de novo protein design is one of the practical

approaches. Therefore our understanding of natural proteins for their folding pathways,

thermodynamic stabilities and catalytic properties is enhanced by our ability to design novel

proteins with predetermined structure and properties. The ability to design proteins de novo

has the potential to bring a revolution in the field of science and technology ranging from

industrial catalyst to biomedical engineering.

Protein design also refers to the effort to design new protein molecules of a desired 3D

structure and function. It is a reverse procedure of protein structure prediction and the

solution of the problem therefore highly relies on the extent of our understanding on the

principle of protein folding.

16. Q. Explain the Strategies used for the designing of protein

structure.

Answer: Strategies used for the designing of protein structure

Finding out an amino acid sequence that will adopt a unique and stable three dimensional

structure is the main goal to design a novel protein. The fundamental hurdle to design a

novel protein is the conformational entropy of the linear polymer chain which must be

overcome. The conformational entropy represents a substantial amount of unfavorable free

energy. For a design to succeed the favorable free energy associated with these designed

interactions must outweigh the entropic cost of fixing the chain into a unique structure.

Many different strategies have been used to achieve this goal. Most of these strategies

have expanded considerable effort to maximize the strength and number of favorable

interactions in the designed structure.The entropic cost of folding is reduced by introducing

covalent cross-links, which limit the number of conformational states accessible to the

chain. Following are the different strategies that have been employed to design novel

protein structure:-

A. Self-Assembly of Modular Units of Secondary Structure

Is the simplest and most direct strategy

In modular approach a single unit of secondary structure i.e. α-structure and β-

structure is synthesized as an individual segment of poly peptide chain.

β-Structures are somewhat less modular than α-structures because individual β-

strands are not stable in isolation they must be linked together by inter-strand

hydrogen bonds.

Self assembly is mediated by non-covalent interactions that are designed into the

peptide.

The key advantage of this approach is its simplicity, both at the level of design and

at the level of synthesis.

The key disadvantage of this approach is the stability of structure and repetitive

structures.

B. Ligand-Induced Assembly

Is the second strategy that uses to design novel protein employ a ligand

Ligand is the metal ion that induces the assembly of modular protein segments.

A ligand binding site is designed into the proposed structure at the interface of

several interacting segments.

If the site have a high affinity for the ligand, then the favorable free energy

associated with binding the ligand will be sufficient to overcome the entropic cost

and drive the peptide to self-assembled.

If the peptide was synthetic in origin, the binding site can be constructed from

moieties other than those represented by the 20 naturally occurring amino acids

side chains.

Metal ion-assisted spontaneous self-assembly of a polypeptide into a triple-helix

bundle protein.

Metal-Directed Protein Self-Assembly, is utilizes the simultaneous stability, lability

and directionality of metal-ligand bonds to drive protein-protein interactions.

The use of metal coordination to control protein self-assembly is attractive from

both structural and functional perspectives: whereas the directionality and

symmetry inherent in metal coordination can govern overall supramolecular

geometry, the resulting interfacial metal centers may potentially offer new

reactivities within biological scaffolds.

Synthetic metal coordinating functionalities have previously been employed for

stabilizing coiled-coil assemblies constructing reactive metal binding sites in protein

interiors and tuning the potentials of redox centers, among others.

It was found that in the absence of added metal

(i) the helical structure are only marginally stable and (ii) this stability was

concentration dependent

C. Assembly of peptides via Covalent cross-linking

As we know that the major obstacle in the designing of a novel protein is the

conformational entropy of the polypeptide chain. When structure are assembled

from several unlinked chains this entropic barrier is all the more difficult to

overcome. Therefore reorganizing the peptides by covalently linking them together

is a powerful strategy to direct the formation of a desired structure.

The disulfide bond is the only bond that is used by the nature to cross-linked for

peptides. If a designed protein is made synthetically, then a variety of other cross-

links are also possible. Example: A novel cross-linker called DAB in their initial design

of Betabellin, an eight-stranded β-barrel intended to fold with a simple up-and-

down topology.

The covalent bond (also termed a peptide bond) has unique properties that make

the protein eminently suited for its role in their structure. The peptide bond has

limited rotational freedom because of the partial double-bond character of the

amide moiety, while the remaining bonds in the polymer main chain can rotate

freely. Therefore, one in every three bonds in the chain is fixed. Because of this

constraint, the polypeptide backbone is significantly more structured than typical

synthetic polymers, such as polyethylenes and polyesters, which lack such rigidifying

elements.

The side chain amino group of lysine or ornithine and the side chain carboxylic acid

groups of Glu or Asp can form peptide bonds with each other or with the N-and C-

termini of the main chain. Such cross-linking leads to branched structures that are

topologicaly quite different from ribosomally produced natural proteins. An

advantage of these structure is that the interchain cross-links constrain the position

of the chains relative to one another, and thereby reduce the entropic cost of

forming a unique structure.

D. Assembly of Peptides on a Synthetic Template

The de novo design of polypeptide sequences with a three-dimensional structure

necessary for many biological functions is limited by the complex folding process, or

‘protein folding problem’. This problem can be bypassed through constructing

protein-like molecules with a ‘built-in’ device for intramolecular folding, that is,

proteins of non-natural chain architecture (template-assembled synthetic proteins,

TASP).

An alternate approach to the template-mediated association of peptide chains is the

use of disulfide bridges to tether peptide modules and induce local folding. This

strategy was utilized in the classical design of some members of the betabellin

family: betadoublet176 and betabellins 14D128 and 15D.

A less coercive approach to assembled proteins involves metal-mediated association

of synthetic modules, whereby strong metal-peptide interactions would drive

assembly. Such an approach necessitates the presence of a metal ligand in each

interacting subunit and further dictates that such complexes be exchange-inert.

Both synthetic peptide assemblies exhibit cooperativity in their denaturation by

guanidium hydrochloride, which is a feature associated with compactly folded

structures.

E. Linear poly peptides that fold into Globular Structure

Is the ultimate goal in the designing of novel structure of proteins three dimensional

structures without the assistance of templates or covalent cross-links.

In which a polypeptide folds into its characteristic and functional three-dimensional

structure from random coil. Each protein exists as an unfolded polypeptide or

random coil when translated from a sequence of mRNA to a linear chain of amino

acids. This polypeptide lacks any developed three-dimensional structure (the left

hand side of the neighboring figure). Amino acids interact with each other to

produce a well-defined three-dimensional structure, the folded protein (the right

hand side of the figure), known as the native state.

The first example of a single-chain polypeptide successfully designed to fold into a

stable globular structure was the α4 structure.

Protein design involves a delicate balance between stabilizing a designed structure

and destabilizing competing structure. The process of designing against alternative

structure is sometimes called “negative design”

F. Protein Design by Binary Pattering of Polar and Non-polar amino Acids

Numerous studies of natural proteins have demonstrated that protein structures

are remarkably tolerant to amino acid substitutions. Thus, many different amino

acids can encode the information necessary to produce a given three dimensional

structure. We have taken advantage of this tolerance to develop a general strategy

for protein design. The strategy—called “the binary code” strategy—is based on the

premise that the appropriate patterning of polar and nonpolar residues can direct a

polypeptide chain to fold into elements of secondary structure, while

simultaneously allowing the burial of nonpolar amino acids in a desired tertiary

structure. A designed binary pattern exploits the periodicities inherent in protein

secondary structure: α-helices have a repeating periodicity of 3.6 residues per turn,

whereas β-strands have an alternating periodicity (Fig. 1). Thus, a binary patterned

sequence designed to form amphipathic α-helices would place a nonpolar residue at

every third or fourth position. In contrast, the binary pattern for an amphipathic β-

strand would alternate between polar and nonpolar residues. In the binary code

strategy, the precise three-dimensional packing of the side chains is not specified a

priori. Therefore, within a library of binary patterned sequences,the identity of the

side chain at each polar and nonpolar position can be varied extensively, thus,

facilitating enormous combinatorial diversity.

17. Why enzyme is called biocatalyst? Explain

Ans. Enzyme are called biocatalyst because of is proteinous nature & since protein

is produced by biological system. It will be denatured at high temp. Speed up

reactions without being changed or used up.They are reusable. Action of enzymes

are specific. Work best at a certain range of pH and temperature.

18. What is a coenzymes?

A large number of enzymes require an additional non protein component called prosthetic group for their efficient activity. Prosthetic group are loosely attached to the enzyme and may be divided into two groups – (a) Metal activators; (b) Cofactors or coenzymes. Coenzymes are organic prosthetic group. Which are generally vitanins. In holoenzymes, the apoenzyme (protein part) determines the specificity while coenzyme (prosthetic group) determines the catalytic functional activity of enzyme

19.What do you mean by cofactor? Explain

Answer:

I. Cofectors are either one or more inorganic ions acts as prosthetic group in enzyme,

such as Fe2+, Mg2+ , Mn2+ or Zn2+

II. Strongly attached with protein part of enzyme (apoenzynme )

III. Not act as carriers of specific functional groups such as methyl groups and acyl

groups

IV. Following are the cofectors with their corresponding enzyme .

20.What do you mean by coenzyme? Explain

Answer:

I. is a complex organic or metalloorganic molecule acts as prosthetic group in enzyme, such as FAD, NAD, Biocytin, Lipoate, Pyridoxal phosphate etc

II. Loosely or transiently attached with protein part of enzyme.III. also act as carriers of specific functional groups such as methyl groups and acyl

groups

21.Differentiate between Cofactor and Coenzyme with example

Cofector Conzyme

I. Cofectors are either one or more I. is a complex organic or metalloorganic

inorganic ions acts as prosthetic

group in enzyme, such as Fe2+,

Mg2+ , Mn2+ or Zn2+

II. Strongly attached with protein part

of enzyme (apoenzynme )

III. Not also act as carriers of specific

functional groups such as methyl

groups and acyl groups

molecule acts as prosthetic group in enzyme, such as FAD, NAD, Biocytin, Lipoate, Pyridoxal phosphate etc

II. Loosely or transiently attached with protein part of enzyme.

III. also act as carriers of specific functional groups such as methyl groups and acyl groups

22.What are active sites? Explain

OR

23.What do you understand by active site of an enzyme?

• Answer: The active site is a specialized region of the protein where the enzyme interacts with the substrate.

• The active site of an enzyme is generally a pocket or cleft that is specialized to recognize specific substrates and catalyze chemical transformations.

• The interactions between the active site and the substrate occur via the same forces that stabilize protein structure:

• hydrophobic interactions,

• electrostatic interactions (charge–charge),

• hydrogen bonding, and

• van der Waals interactions.

• It also contains the groups that directly participate in the making and breaking of bonds. These groups are called the catalytic groups.

• Catalytic groups facilitate the chemistry and provide specific interactions that stabilize the formation of the transition state for the chemical reaction.

• Therefore the interaction of the enzyme and substrate at the active site promotes the formation of the transition state.

• The active site is the region of the enzyme that most directly lowers the activation energy of the reaction, which results in the rate enhancement characteristic of enzyme action.

Two models have been proposed to explain how an enzyme binds its substrate. In the lock-and-key model proposed by Emil Fischer in 1894, the shape of the substrate and the active site of the enzyme are thought to fit together like a key into its lock (Fig. 1a). The two shapes are considered as rigid and fixed, and perfectly complement each other when brought together in the right alignment. In the induced-fit model proposed in 1958 by Daniel E. Koshland, Jr., the binding of substrate induces a conformational change in the active site of the enzyme (Fig. 1b). In addition, the enzyme may distort the substrate, forcing it into a conformation similar to that of the transition state. For example, the binding of glucose to hexokinase induces a conformational change in the structure of the enzyme such that the active site assumes a shape that is complementary to the substrate (glucose) only after it has bound to the enzyme. Different enzymes show features of both models, with some complementarity and some conformational change.

24.Define Km and Vmax.

Answer: Km (Michaelis Mention Constant) is the substrate concentration at which the

chemical reaction attains half its maximum velocity. o Km is the concentration of substrate required to achieve 1/2 Vmax.o It is an inverse measure of the affinity of an enzyme for its substrate. Small the

Km, the greater the substrate affinity. o Allosteric enzymes do not obey Km constant.o Km is a measure of the stability of the ES complex, being equal to the sum of

the rates of breakdown of ES over its rate of formation. o Km is a measure of the affinity of an enzyme for its substrate .o A high Km indicates weak substrate binding , a low Km indicates strong

substrate binding . o Km can be determined experimentally by the fact that its value is equivalent to

the substrate concentration at which the velocity is equal to half of Vmax. Vmax is velocity of enzyme catalyze reaction at maximum or saturated substrate

concentration.

o Km = Vmax / 2

o Vmax = kcat, x [Enz]

Fig: The relationship between substrate concentration [S] and initial reaction velocity (V0 ). (a) A direct plot, (b) a Lineweaver–Burk double-reciprocal plot.

25.What is significance of Km, Vmax, Vmax / Km?

Answer: Km is the concentration of substrate required to achieve 1/2 Vmax.o It is an inverse measure of the affinity of an enzyme for its substrate. Small the

Km, the greater the substrate affinity. o Allosteric enzymes do not obey Km constant.o Km is a measure of the stability of the ES complex, being equal to the sum of

the rates of breakdown of ES over its rate of formation. o Km is a measure of the affinity of an enzyme for its substrate .o A high Km indicates weak substrate binding , a low Km indicates strong

substrate binding .

o Km can be determined experimentally by the fact that its value is equivalent to

the substrate concentration at which the velocity is equal to half of Vmax.

o Km and Vmax have different meanings for different enzymes. o The limiting rate of an enzyme-catalyzed reaction at saturation is described by

the constant kcat, also known as the turnover number. o Turnover number is equivalent to the number of substrate molecules converted

to product in a given unit of time on a single enzyme molecule when the enzyme is saturated with substrate.

o The ratio kcat/Km provides a good measure of catalytic efficiency.

26.What do you mean by enzyme ‘s Kinetic parameters? Explain

The kinetic parameters kcat and Km are generally useful for the study and comparisonof different enzymes, whether their reaction mechanisms are simple or complex. Eachenzyme has values of kcat and Km that reflect the cellular environment, the concentration of substrate normally encountered in vivo by the enzyme, and the chemistry of the reaction being catalyzed.The parameters kcat and Km also allow us to evaluate the kinetic efficiency of enzymes, but either parameter alone is insufficient for this task. Two enzymes catalyzing different reactions may have the same kcat (turnover number), yet the rates of the uncatalyzed reactions may be different and thus the rate enhancements brought about by the enzymes may differ greatly. Experimentally, the Km for an enzyme tends to be similar to the cellular concentrationof its substrate. An enzyme that acts on a substrate present at a very low concentration in the cell usually has a lower Km than an enzyme that acts on a substrate that is more abundant. The best way to compare the catalytic efficiencies of different enzymes or the turnover of different substrates by the same enzyme is to compare the ratio kcat/Km for the two reactions. This parameter, sometimes called the specificity constant, is the rate constant for the conversion of E +S to E + P.

When [S] << Km, then

V0 in this case depends on the concentration of two reactants, [Et] and [S]; therefore this is a second-order rate equation and the constant kcat/Km is a second-order rate constant with units of M-1s-1. There is an upper limit to kcat/Km, imposed by the rate at which E and S can diffuse together in an aqueous solution. This diffusion controlled limit is 108 to 109 M-1s-1, and many enzymes have a kcat/Km near this range given in the table below. Such enzymes are said to have achieved catalytic perfection. Note that different values of kcat and Km can produce the maximum ratio.

27.State two important properties of enzymes briefly.

Answer:

1. ENZYMES ARE POWERFUL AND HIGHLY SPECIFIC CATALYSTSEnzymes accelerate reactions by factors of as much as a million or more Indeed, most reactions in biological systems do not take place at perceptible rates in the absence of enzymes. Even a reaction as simple as the hydration of carbon dioxide is catalyzed by an enzyme—namely, carbonic anhydrase. The transfer of CO2 from the tissues into the blood and then to the alveolar air would be less complete in the absence of this enzyme. In fact, carbonic anhydrase is one of the fastest enzymes known. Each enzyme molecule can hydrate 106 molecules of CO2 per second. This catalyzed reaction is 107 times as fast as the uncatalyzed one. Enzymes are highly specific both in the reactions that they catalyze and in their choice of reactants, which are called substrates. An enzyme usually catalyzes a single chemical reaction or a set of closely related reactions. Side reactions leading to the wasteful formation of by-products are rare in enzyme-catalyzed reactions, in contrast with uncatalyzed ones.

The specificity of an enzyme is due to the precise interaction of the substrate with the enzyme. This precision is a result of the intricate three-dimensional structure of the enzyme protein.

2. Enzymes Affect Reaction Rates, Not Equilibria:

The function of a enzymes / catalyst is to increase the rate of a reaction by lowering the activation energy, ∆G‡, for a reaction and thereby enhance the reaction rate. The equilibrium of a reaction is unaffected by the enzyme.

3. Many Enzymes Require Cofactors for Activity:The catalytic activity of many enzymes depends on the presence of small molecules termed cofactors, although the precise role varies with the cofactor and the enzyme. Such an enzyme without its cofactor is referred to as an apoenzyme; the complete, catalytically active enzyme is called a holoenzyme. Cofactors can be subdivided into two groups: metals and small organic molecules . The enzyme carbonic anhydrase, for example, requires Zn2+

for its activity. Cofactors that are small organic molecules are called coenzymes. Often derived from vitamins, coenzymes can be either tightly or loosely bound to the enzyme. If tightly bound, they are called prosthetic groups. Loosely associated coenzymes are more like cosubstrates because they bind to and are released from the enzyme just as substrates and products are. The use of the same coenzyme by a variety of enzymes and their source in vitamins sets coenzymes apart from normal substrates, however. Enzymes that use the same coenzyme are usually mechanistically similar.

28.What are the six main classes of enzyme? Explain

Answer: The enzymes are classified into 6 groups on the basis of type of reactionsthey catalyse:(a) Oxidoreductases: Transfer of H and O atoms or electrons from one substanceto another. Examples are dehydrogenase, oxidase.

(b) Transferases: Transfer of a specific group (methyl, aceyl, amino or phosphate)from one substance to another. Examples: transaminase, kinase.

(c) Hydrolases: Hydrolysis of a substrate. Examples are lipase, amylase, peptidase,esterase, phosphatase, carbohydrase, and protease.

(d) Lyases: Non hydrolytic removal or addition of group from substrates, C–C, C–N, C–O, or C–S bonds may be split. Examples are decarboxylase, fumarase, andaldolase.

(e) Isomerases: Change of a substrate into a related form by intramolecularrearrangement. Examples are phosphohexose isomerase.

(f) Ligases (synthetases): Joining of two molecules by synthesis of new C–O, C–

S, C–N or C–C bonds with simultaneous breakdown of ATP. Examples are acetylCoA synthetase (acting on fatty acids), pyruvate carboxylase.

29.Write down the examples of coenzyme.

Answer: The proteinaceous part of enzyme is called apoenzyme. The apoenzyme plus non proteinaceous part is called holoenzyme. Some enzymes require a loose association with certain organic substances for their activity. These prosthetic groups are called cofactors or coenzymes. Examples of coenzymes are – NAD (nicotinamide adenine dinucleotide), NADP (nicotinamide adenine dinucleotide phosphate), ATP (adenosine triphosphate), CoA (coenzyme A), FMN (Flavin mononucleotide) and FAD (Flavin adenine dinucleotide). FAD and FMN contain riboflavin (vitamin B2) as a component. Riboflavin is the hydrogen accepting part of FAD/FMN.

30.Give the introduction of Enzyme?

ANS: 1. Enzymes are also known as biological catalyst was discovered by Buchner (1897-1903) but term enzyme was given by Kuhne (1878).2. Almost all enzymes are proteins. However, ribozyme, ribonuclease P are nonprotein enzyme.3. Every cell produces its own enzymes because they cannot move from cell to cell dueto high molecular weight.4. All components of cell including cell wall and cell membrane have enzymes.5. Maximum enzymes in the cell are found in mitochondrion.6. Smallest enzyme is peroxidase and largest enzyme being catalase found inperoxisomes.7. Enzyme urease isolated from Jack bean Canavalia was crystallized by Summer in1926, who proved protein nature of enzymes.

8. Enzymes show reversible reactions and act by lowering energy of activation by morethan 50%.9. Enzymes show three dimensional structures.10. Over 2000 enzymes have been recorded. Enzymes are synthesized by living cells.Most of the enzymes remain and function inside the cells. These are calledendoenzymes (or intracellular enzymes). On the other hand, the enzymes whichleave the cells and function outside them are called exoenzymes (or extracellularenzymes). These retain their catalytic ability even when extracted from cells. Rennettablets (containing the enzyme rennin from the calf’s stomach) have been in use for

coagulating milk protein to obtain casein (cheese from milk).11. Enzymes are required in minute quantities which is sufficient to convert a largeamount of substrates (starting materials of a reaction) to products (ending materialsof a reaction).12. Enzymes are generally specific for the type of reactions they catalyse. This specificityis very strong for some enzymes.

13. Enzymes are colloidal in nature; and have high molecular weights raning from 10,000–50,000. However, the molecular weights of catalase and urease are 2,50,000 and4,83,000 respectively.14. The enzyme lowers the activation energy of a reaction. (The energy required forsubstrates to react in order to get converted into product is called energy of activation).An enzyme (E) combines with its substrates (S) to form a short-lived enzyme-substrate(ES) complex. Within this complex, the chances of occurring of reaction are greatlyincreased. Once a reaction has occurred, the complex breaks up into products andenzymes. Thus, the enzyme remains unchanged at the end of the reaction; and isfree to interact again with more substrates.

31. What do you mean by prosthetic group of enzyme?ANS: A large number of enzymes require an additional non protein component called prosthetic group for their efficient activity. Prosthetic group may be divided rather loosely into two groups – (a) Metal activators; (b) Cofactors or coenzymes.In holoenzymes, the apoenzyme (protein part) determines the specificity while coenzyme (prosthetic group) determines the catalytic functional activity of enzyme. The proteinaceous part of enzyme is called apoenzyme. The apoenzyme plus non proteinaceous part is called holoenzyme. Some enzymes require a loose association with certain organic substances for their activity. These prosthetic groups are called cofactors or coenzymes. Examples of coenzymes are – NAD (nicotinamide adenine dinucleotide), NADP (nicotinamide adenine dinucleotide phosphate), ATP (adenosine triphosphate), CoA (coenzyme A), FMN (Flavin mononucleotide) and FAD (Flavin adenine dinucleotide). FAD and FMN contain riboflavin (vitamin B2) as a component. Riboflavin is the hydrogen accepting part of FAD/FMN.

32.Explain the different components or part of enzyme.

ANS: A large number of enzymes require an additional non protein component called prosthetic group for their efficient activity. Prosthetic group may be divided rather loosely into two groups – (a) Metal activators; (b) Cofactors or coenzymes. In holoenzymes, the apoenzyme (protein part) determines the specificity while coenzyme (prosthetic group) determines the catalytic functional activity of enzyme.The proteinaceous part of enzyme is called apoenzyme. The apoenzyme plus nonproteinaceous part is called holoenzyme. Some enzymes require a loose association with certain organic substances for their activity. These prosthetic groups are called cofactors or coenzymes. Examples of coenzymes are – NAD (nicotinamide adenine dinucleotide),

NADP (nicotinamide adenine dinucleotide phosphate), ATP (adenosine triphosphate), CoA (coenzyme A), FMN (Flavin mononucleotide) and FAD (Flavin adenine dinucleotide). FAD and FMN contain riboflavin (vitamin B2) as a component. Riboflavin is the hydrogen accepting part of FAD/FMN.

33.How pH and Temprature affect the enzyme?

ANS: Every enzyme has its own optimum pH. Any shift towards alkaline or acidic side results in a decrease in enzyme activity because it denatures the enzyme molecule (changes its shape). Pepsin of gastric juice has optimum activity at pH 2.0, while trypsin shows maximum acitivity at pH 8.0.Every enzyme has a specific optimum temperature. Over a range of 0–40°C, the rate of enzyme controlled reaction almost doubles for every rise of 10°C. (Q10 = 2).Most enzymes show maximum activity in a temperature range of 25–40°C.Enzymes are thermolabile i.e. are denatured at high temperatures. The loss of catalytic properties begins at 36°C and is almost complete as 60°C is reached. However, dried enzyme extracts can endure temperature of 100°C–120°C or even higher. That is why; dry seeds can endure high temperature than germinating seeds.

34.What are the classifications of enzyme?

IUB (1962) has divided enzymes into 6 classes (oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases). Each class is divided into sub-classes and each sub-class into sub-sub classes depending upon the type of reaction and nature of substrate. Thus every enzyme has a four digit code called EC Number (Enzyme Commission Number).The enzymes are classified into 6 groups on the basis of type of reactions they catalyse:(a) Oxidoreductases: Transfer of H and O atoms or electrons from one substance to another. Examples are dehydrogenase, oxidase.(b) Transferases: Transfer of a specific group (methyl, aceyl, amino or phosphate) from one substance to another. Examples: transaminase, kinase.(c) Hydrolases: Hydrolysis of a substrate. Examples are lipase, amylase, peptidase, esterase, phosphatase, carbohydrase, and protease.(d) Lyases: Non hydrolytic removal or addition of group from substrates, C–C, C– N, C–O, or C–S bonds may be split. Examples are decarboxylase, fumarase, and aldolase.(e) Isomerases: Change of a substrate into a related form by intramolecular rearrangement. Examples are phosphohexose isomerase.(f) Ligases (synthetases): Joining of two molecules by synthesis of new C–O, C– S, C–N or C–C bonds with simultaneous breakdown of ATP. Examples are acetyl CoA synthetase (acting on fatty acids), pyruvate carboxylase.

35.Write short notes on active site.

Most enzymes are far larger molecules than substrates. Only a small portion of the enzyme (3-12 amino acids) comes into direct contact with the substrate. This region is called the active site of the enzyme. An enzyme may have more than one active site. The remaining amino acids maintain the correct globular shape of the molecule. It is important for proper functioning of the active site.Fischer (1890) proposed lock and key hypothesis to explain specificity. He proposed that enzymes have a particular shape into which the substrate or substrates fit exactly. For a lock to work, it must be provided with right key. Similar is the case with enzyme and substrates.The best evidence for this lock and key hypothesis (or template theory) of enzyme action comes from the observation that compounds similar in structure to the substrate inhibit the reaction.Evidence from protein chemistry suggested that a slight rearrangement of chemical groups occurs in both enzyme and substrate when as ES complex is formed. It means that enzymes and their active sites are rather flexible structures. Koshland (1959),therefore, suggested induced-fit hypothesis. According to it, when a substrate combines with an enzyme, it induces change in the enzyme structure. The amino acids constituting the active site are moulded into a precise formation which enables the enzyme to perform its catalytic function more effectively.

36.Define the following terms. Alloenzymes, Constitutive enzymes, Repressible Inducible enzymes enzymes, ELISA and Restiction endonuclease

ANS: • Alloenzymes are enzymes which are produced by different genes.• Constitutive enzymes are always present because they are always required for vital process e.g. glycolysis.• Repressible enzymes – normally remain present but are repressed when a specific chemical or product is present e.g. glucokinase.• Inducible enzymes – are formed in response to presence of its substrate e.g. lactose.• ELISA – It is an enzyme linked immunosorbentassay when a protein, antibody or antigen is detected by means of a specific enzyme e.g. AIDS.• Restiction endonuclease – These are enzymes which are used to break DNA ata specific site producing sticky ends. The enzymes are highly important for genetic engineering. Arber, Nathans and Smith were awarded Nobel Prize in 1978 for their discovery.

37.What do you mean by quantitation of enzymes?

1. activity = “how much” ( moles product formed per minute)2. specific activity = “how pure” ( moles product/min. per mg protein)3. Turnover number, kcat = “how efficient” = Vmax/[ET] (moles product/min.per mole enzyme)

38.What are the factors influencing enzyme activity?

A. Environmental factors1. Temperature2. Ionic strength3. pH4. Concentrations of substrates (reactants), products, cofactorsB. Rate of synthesis and degradation of enzyme (minutes to hours)C. Isozymes: multiple forms of an enzyme catalyzing the same reaction in thesame organismD. Covalent modification of enzyme (seconds to minutes)E. Allosteric regulation (seconds)F. Proteolytic cleavage:1. Activation: zymogens are inactive enzyme precursors that are activated byproteolytic cleavage (e.g. digestive enzymes trypsin, chymotrypsin, etc.)2. Inactivation by proteolysis: also digestive enzymesG. Binding of a regulatory protein: e.g. trypsin and trypsin inhibitorH. Compartmentation:1. Metabolic channeling: direct transfer of substrate to next enzyme (Pyruvatedehydrogenase complex)2. Separate subcellular location of catabolic, anabolic pathways (e.g. fatty acidoxidation in mitochodria, synthesis in cytosol)

58 Briefly explain the mechanism of action of enzyme.

MECHANISMS OF ACTION ENZYMES can be explain by following theoretical explanations for enzyme catalysisA. “Lock and Key”1. Proximity - “local concentration”2. Orientation effects - alignment of bonds to facilitate catalysisB. Induced fit - Hexokinase (enzyme conforms to substrate)C. Induced strain - Lysozyme (substrate conforms to enzyme )D. Transition state stabilization (TSS)1. Transition state analogs are excellent enzyme inhibitors2. Drug design based on Transition State analogs3. Abzymes: antibodies against transition state analogs have enzymatic activity

IV. Enzymatic catalysis mechanismsA. Acid-base catalysis1. General model Enz–H+ + R'– OR → Enz + R'– H + R+R+ + H2O + Enz → Enz–H + R – O –H2. Specific examples: (a) Lysozyme (b) Ribonuclease

B. Covalent catalysis: intermediate with covalent bond between E and S1. General model: Enz–O– H + A–B → Enz–O–A + B– H

Enz–O–A + H2O → Enz – O – H + A – O –H2. Specific examples:(a) Chymotrypsinb) Subtilisin: convergent evolution

C. Metal ion catalysis1. Function as Lewis acids2. Function to stabilize intermediates (chelates)3. Facilitate binding by neutralizing charges4. Involved in many redox reactions

59. Explain the energetics of enzyme-substrate complex formation.Prominent physical and thermodynamic factors contributing to activation energy, the barrier to reaction, might include-

(1) a reduction in entropy, in the form of decreased freedom of motion of two molecules in solution;

(2) the solvation shell of hydrogen-bonded water that surrounds and helps to stabilize most biomolecules in aqueous solution;

(3) the distortion of substrates that must occur in many reactions; and

(4) the need for proper alignment of catalytic functional groups on the enzyme.

Binding energy release during binding of enzyme with their substrate can be used to overcome all the barriers and lower the activation energy

60.What do you mean by active site?

• The active site is a specialized region of the protein where the enzyme interacts with the substrate.

• The active site of an enzyme is generally a pocket or cleft that is specialized to recognize specific substrates and catalyze chemical transformations.

• The interactions between the active site and the substrate occur via the same forces that stabilize protein structure:

• hydrophobic interactions, • electrostatic interactions (charge–charge), • hydrogen bonding, and • van der Waals interactions.

• It also contains the groups that directly participate in the making and breaking of bonds. These groups are called the catalytic groups.

• Catalytic groups facilitate the chemistry and provide specific interactions that stabilize the formation of the transition state for the chemical reaction.

• Therefore the interaction of the enzyme and substrate at the active site promotes the formation of the transition state.

• The active site is the region of the enzyme that most directly lowers the activation energy of the reaction, which results in the rate enhancement characteristic of enzyme action.

61.What are the characteristics of enzyme?

The characteristics of enzyme are:

• Protein in nature & will be denatured at high temp.

• Biological catalysts . Speed up reactions without being changed or used up.

• They are reusable.

• Action of enzymes are specific.

• High selectivity (enantio-, regio-, chemo-)

• Mild reaction conditions and energy efficient

• High turnover numbers and enormous rate enhancements

• Reduced byproduct yield

• Accepts complex substrates

• Environmental friendliness

• Derived from natural sources and completely degraded in the environment

• “Self-contained”, renewable catalysts

• Work best at a certain range of pH and temperature.

62. What are different type of Enzyme selectivity?

Ans. There are four type of enzyme selectivity. These are:

1. Functional selectivityi. Types of reactions catalyzed

ii. Not unique to enzymesiii. Potential for reduced byproduct yields

2. Substrate selectivityi. Specificity toward a subset of reactants

ii. Enzymes are nearly unique3. Enantioselectivity

i. Chirality ii. Enzymes have major advantage over chemical catalysts

4. Regioselectivity i. Positional specificity

ii. Enzymes have major advantage over chemical catalysts

63. What are the characteristic of active site? Explain.

Ans. Characteristics of Active Site:

• Although enzymes differ widely in structure, specificity, and mode of catalysis, a

number of common feature found in active site of enzyme which are :

• 1.The active site is a three-dimensional cleft formed by groups that come from

different parts of the amino acid sequence.

• 2. The active site takes up a relatively small part of the total volume of an

enzyme. Nearly all enzymes are made up of more than 100 amino acid

residues, which gives them a mass greater than 10 kd and a diameter of more

than 25 Å. The “extra” amino acids serve as a scaffold to create the three

dimensional active site from amino acids that are far apart in the primary

structure.

• 3. Active sites are clefts or crevices. In all enzymes of known structure,

substrate molecules are bound to a cleft or crevice. Contain both polar and

non polar group like –NH2, -COOH, -SH, -OH, -CH3 for establishing the contact

with the substrate molecules.

• 4. Substrates are bound to enzymes by multiple weak attractions like

electrostatic interactions, hydrogen bonds, van der Waals forces, and

hydrophobic interactions mediate reversible interactions

• 5. The specificity of binding depends on the precisely defined arrangement of

atoms in an active site. Because the enzyme and the substrate interact by

means of short-range forces that require close contact, a substrate must have

a matching shape to fit into the site like lock and key model and Induced-fit

model

64.What do you understand by feed-back inhibition? Explain

Answer: In most multienzyme systems, the first enzyme of the sequence is a regulatory enzyme. In some multienzyme systems, the regulatory enzyme is specifically inhibited by the end product of the pathway whenever the concentration of the end product exceeds the cell’s requirements. When the regulatory enzyme reaction is slowed, all subsequent enzymes operate at reduced rates as their substrates are depleted. The rate of production of the pathway’s end product is thereby brought into balance with the cell’s needs. This type of regulation is called feedback inhibition or allosteric inhibition or allosteric regulation. Buildup of the end product ultimately slows the entire pathway.

Example of allosteric enzyme

One of the first known examples of allosteric feedback inhibition was the bacterial enzyme system that catalyzes the conversion of L-threonine to L-isoleucine in five steps shown in the figure. In this system, the first enzyme, threonine dehydratase, is inhibited by isoleucine, the product of the last reaction of the series. This is an example of heterotropic allosteric inhibition. Isoleucine is quite specific as an inhibitor. No other intermediate in this sequence inhibits threonine dehydratase, nor is any other enzyme in the sequence inhibited byisoleucine. Isoleucine binds not to the active site but to another specific site on the enzyme molecule, the regulatory site. This binding is noncovalent and readily reversible; if the isoleucine concentration decreases, the rate of threonine dehydration increases. Thus threonine dehydratase activity responds rapidly and reversibly to fluctuations in the cellular concentration of isoleucine.

The conversion of L-threonine toL-isoleucine is catalyzed by a sequence of five enzymes (E1 to E5).Threonine dehydratase (E1) is specifically inhibited allosterically byL- isoleucine, the end product of the sequence, but not by any of the four intermediates (A to D).

68 .What are allosteric enzymes?

Answer: An important group of enzymes that do not obey Michaelis- Menten kinetics.They have allosteric site other than active site. These enzymes consist of multiple subunits and multiple active sites. Allosteric enzymes often display sigmoidal plots of the reaction velocity V0 versus substrate concentration [S], rather than.

70. Discuss briefly allosteric enzymes. Answer: An important group of enzymes that do not obey Michaelis- Menten kinetics. They have allosteric site other than active site. These enzymes consist of multiple subunits and multiple active sites.

Allosteric enzymes often display sigmoidal plots of the reaction velocity V0 versus substrate concentration [S], rather than.

In allosteric enzymes, the binding of substrate to one active site can affect the properties of other active sites in the same enzyme molecule. The binding of substrate to one active site of the enzyme facilitates substrate binding to the other active sites is called cooperativity . Such cooperativity results in a sigmoidal plot of V0 versus [S]. In addition, the activity of an allosteric enzyme may be altered by regulatory molecules that are reversibly bound to specific sites other than the catalytic sites. The catalytic properties of allosteric enzymes can thus be adjusted to meet the immediate needs of a cell . For this reason, allosteric enzymes are key regulators of metabolic pathways in the cell.

71 .Discuss briefly allosteric enzymes with one suitable example.

Answer: Refer to answer of question no. 28

72 How do you differentiate between Michaelian and allosteric enzyme? Give one

example of allosteric enzyme.

Answer:

Michaelian or Michaelis- Menten

Enzyme

Allosteric Enzyme

Obey Michaelis- Menten kinetics An important group of enzymes that do not obey Michaelis- Menten kinetics.

These enzymes consist of single active sites

Having no allosteric site

These enzymes shows the hyperbolicplots drawn by the Michaelis-Menten equation.

It is not happens.

Cooperativity is not shown by this enzyme.

This enzymes are generally not key regulators of metabolic pathways in the cell.

These enzymes consist of multiple subunits and multiple active sites.

Having allosteric site other than active site

Allosteric enzymes often display sigmoidal plots of the reaction velocity V0 versus substrate concentration [S], rather than.

In allosteric enzymes, the binding of substrate to one active site can affect theproperties of other active sites in the same enzyme molecule.

The binding of substrate to one active site of the enzyme facilitates substrate binding to the other active sites is called cooperativity . Such cooperativity results in a sigmoidal plot of V0 versus [S].

In addition, the activity of an allosteric enzyme may be altered by regulatory molecules that are reversibly bound to specific sites other than the catalytic sites. The catalytic properties of allosteric enzymes can thus be adjusted to meet the immediate needs of a cell . For this reason, allosteric enzymes are key regulators of metabolic pathways in the cell.

Example of allosteric enzyme

One of the first known examples of allosteric feedback inhibition was the bacterial enzyme system that catalyzes the conversion of L-threonine to L-isoleucine in five steps shown in the figure. In this system, the first enzyme, threonine dehydratase, is inhibited by isoleucine, the product of the last reaction of the series. This is an example of heterotropic allosteric inhibition. Isoleucine is quite specific as an inhibitor. No other intermediate in this sequence inhibits threonine dehydratase, nor is any other enzyme in the sequence inhibited by

isoleucine. Isoleucine binds not to the active site but to another specific site on the enzyme molecule, the regulatory site. This binding is noncovalent and readily reversible; if the isoleucine concentration decreases, the rate of threonine dehydration increases. Thus threonine dehydratase activity responds rapidly and reversibly to fluctuations in the cellular concentration of isoleucine.

The conversion of L-threonine toL-isoleucine is catalyzed by a sequence of five enzymes (E1 to E5).Threonine dehydratase (E1) is specifically inhibited allosterically byL-isoleucine, the end product of the sequence, but not by any of thefour intermediates (A to D).

Unit II

73. Demostrate the procedure of immobilization of enzyme in a flow chart.

Answer:

73 . What are the Ideal Attributes of an Enzyme Carrier?

High surface area, Permeable, Insoluble, High rigidity, Regenerable, Resistant

to microbial and chemical attack, Mechanically and thermally stable and

Controlled hydrophobicity

74 . What are the key immobilization methods?

Answer:

75 . Schematically represent the different immobilization methods

Note: a. Adsorption b. Covalent attachement c. enzyme entrapment d.

encapsulation

76 Explain the application of immobililized enzyme.

Applications:

77. What is enzyme immobilization?

Answer: The process of enzyme immobilization is physically confining or localizing the biocatalyst in a certain defined region of space with retention of its catalytic activity, which can be used repeatedly and continuously.The objective of immobilization is the economic application of enzyme systems. Other benefits such as ease of control and uniformity of conversions may be derived from immobilization techniques. The greatest return from immobilization is achieved with expensive enzymes since there is a definitive cost associated with immobilizing processes. Hence enzymes that are more expensive to produce, including many intracellular microbial enzymes, and those employed in biosensors and analytical test systems, are often used in an immobilized form.It is clearly apparent. Then, that a very inexpensive enzyme should not be employed as an immobilized derivative in a process unless another advantage such as avoidance of contamination or immune response is attained.

78.How enzyme immobilization is carried out ?

There are five markedly different techniques of immobilizing enzymes:1. adsorption of the enzyme on a carrier surface2. ionic bonding or covalent coupling of an enzyme to a carrier surface ie. inert inorganic or organic solid support ma-terials, such as nylon, bentonite, cellulose and dextran.3. Cross linking between enzyme molecules (copolymerization);4. entrapment of an enzyme in a matrix ie. within gels or fibres, and intracellular enzymes may be immobilized within their producer cells.5. encapsulation or confinement of an enzyme solution in a membrane structure.Although each immobilization technique is unique, they are by no means pure processes and are in combination with other methods.· Adsorption on a car-rier surface, either intentionally or unintentionally, will involve crosslinking between enzyme molecules to some extent.· Covalent coupling to a carrier surface usually involves adsorption of enzymes on that surface and crosslinking between enzyme molecules.· Entrapment of an enzyme in a matrix may involve adsorption, covalent coupling to the surface or crosslinking between the enzyme molecules.· Encapsulation or confinement of an enzyme solution within a membrane structure minimizes contamination by these mixed immobilization effects, however, someadsorption on the membrane surface and some crosslink king of the molecules do occur.Each immobilization process has both advantages and disadvantages. The choice of technique, therefore, should be decided by the specific con-ditions of the application which would selectively employ the positive attributes of the specified immobilization.

79. What is enzyme immobilization?

Answer: The process of enzyme immobilization is physically confining or localizing the biocatalyst in a certain defined region of space with retention of its catalytic activity, which can be used repeatedly and continuously.The objective of immobilization is the economic application of enzyme systems. Other

benefits such as ease of control and uniformity of conversions may be derived from

immobilization tech-niques. The greatest return from immobilization is achieved with

expensive enzymes since there is a definitive cost associated with immobilizing processes.

Hence enzymes that are more expensive to produce, includ-ing many intracellular microbial

enzymes, and those employed in biosensors and analytical test systems, are often used in an

immobilized form.

80.What are advantages and disadvantages of enzyme immobilization?

81.How Immobilized enzymes are very important for commercial uses

Answer: Immobilized enzymes are very important for commercial uses as they possess many benefits to the expenses and processes of the reaction of which include:

Convenience: Minuscule amounts of protein dissolve in the reaction, so workup can be much easier. Upon completion, reaction mixtures typically contain only solvent and reaction products.

Economical: The immobilized enzyme is easily removed from the reaction making it easy to recycle the biocatalyst.

Stability: Immobilized enzymes typically have greater thermal and operational stability than the soluble form of the enzyme.

82.Compare the different immobilization method.

Answer:

83.Shown a graph for effect of immobilization on stability of enzyme.

84.Write short notes on Immobilization by Entrapment.

Gel entrapment places the enzyme within the interstitial spaces of crosslinked, water-

insoluble polymer gels. Polysaccharides: The solubility of alginate and k-Carrageenan

varies with the cation, allowing these soluble polymers to be crosslinked upon the addition

of CaCl2 and KCl, respectively. Variations of pore size result in enzyme leakage, even after

washing. The effect of initiator used in polyacrylamide gels can be problematic.

Microencapsulation encloses enzymes within spherical, semi-permeable membranes of 1-

100 mm diameter.

Urethane prepolymers, when mixed with an aqueous enzyme solution crosslink via urea

bonds to generate membranes of varying hydrophilicity.

Alternatively, photo- crosslinkable resins can be gelled by UV-irradiation.

Advantage of Entrapment

Enzymes are immobilized without a chemical or structural modification. A

very general technique.

Disadvantage of Entrapment

High molecular weight substrates have limited diffusivity, and cannot be

treated with entrapped enzymes.

85.What do you mean by Immobilization by Carrier Binding.

Attachment of an enzyme to an insoluble carrier creates an active surface catalyst. Modes

of surface attachment classify carrier methods into physical adsorption, ionic binding and

covalent binding.

Physical Adsorption: Enzymes can be bound to carriers by physical interaction such as

hydrogen bonding and/or van der Waal’s forces.

the enzyme structure is unmodified

carriers include chitosan, acrylamide polymers and silica-alumina

binding strength is usually weak and affected by temperature and the

concentration of reactants.

Ionic Binding: Stronger enzyme-carrier binding is obtained with solid supports containing

ion-exchange residues.

cellulose, glass-fibre paper, polystyrene sulfonate

pH and ionic strength effects can be significant

Covalent attachment of soluble enzymes to an insoluble support is the most common

immobilization technique.

Amino acid residues not involved in the active site can be used fix the enzyme

to a solid carrier

Advantages:

1. Minimal enzyme leaching from the support results

in stable productivity

2. Surface placement permits enzyme contact with

large substrates

Disadvantages:

1. Partial modification of residues that constitute the active site

decreases activity

2. Immobilization conditions can be difficult to optimize (often done

in the presence of a competitive inhibitor)

86.What do you mean by Immobilization by Crosslinking.

Bi- or multi-functional compounds serve as reagents for intermolecular crosslinking of

enzymes, creating insoluble aggregates that are effective heterogeneous catalysts.

Reagents commonly have two identical functional groups which react with specific amino

acid residues.Common reagents include glutaraldehyde, and diisocyanates,

Involvement of the active site in crosslinking can lead to great reductions in activity, and

the gelatinous nature of the product can complicate processing.

87.What will be the effect of Immobilization on Operational Stability

Answer: Given that activity of enzymes is dictated by structure and conformation, the

environmental change resulting from immobilization affects not only maximum activity,

but the stability of the enzyme preparation.

The factors that inactivate enzymes are not systematically understood, and

depend on the intrinsic nature of the enzyme, the method of immobilization,

and the reaction conditions employed.

In general, immobilized enzyme preparations demonstrate better stability.

Note that the immobilized preparation is often more stable than the soluble enzyme and

displays a period during which no enzyme activity appears to be lost.

Note 1 represent mmobilized enzyme

2 represent free soluble enzyme

88.Give important applications of enzyme immobilization in industries.

Answer:

89.What do you mean by enzyme reactor?

Answer: A vessel employed to carry out the desired conversion using an enzyme is called

enzyme reactor. Several different types of reactors are available, the choice of a reactor

type depending on the form of enzyme (free or immobilized) to be used, kinetics of

reaction, etc., and the scale of operation.

The different types of reactors used for enzyme-mediated .conversions are as follows:

(i) stirred tank reactors,

(ii) membrane reactors,

(iii) continuous flow reactors, e.g.,

(iv) packed bed reactors,

90. What do you mean by STR?

Answer: A enzyme reactor in which an immobilized enzyme tends to decompose or work upon physical stirring. These reactors are simple and consist of a tank containing a stirrer and, usually, fixed baffles to improve mixing. These reactors are used in batch mode,

and free enzymes can be employed. Efficient arrangements are provided to maintain temperature, pH, etc. of the reaction mix.

91.What do you mean by fluidized bed reactor?

In fluidized bed reactors, cells are "immobilized" small particles which move with the fluid. The small particles create a large surface area for cells to stick to and enablea high rate of transfer of oxygen and nutrients to the cells

92.What do you mean by packed bed reactor?

In packed bed reactors, cells are immobilized on large particles. These particles do not move with the liquid. Packed bed reactors are simple to construct and operatebut can suffer from blockages and from poor oxygen transfer.

93.What do you mean by membrane reactor?

Answer: A membrane reactor uses a membrane, e.g., dialysis membrane, to

contain the enzyme in a chamber into which the substrate moves and the

product moves out. Generally, membrane reactors use a hollow fiber of 200

/lm diameter with 50 /lm thick membranes.

94.What is different type of enzyme reactors?

Answer:

- An immobilized enzyme tends to decompos upon physical stirring.

- The batch system is generally suitable for the production of rather small amounts

of chemicals.

Fluidized Bed Reactor:- a high viscosity substrate solution a gaseous substrate or product in a continuous reaction system care must be taken to avoid the destruction and

decomposition of immobilized enzymes

95.What are the parameter affecting the performance of enzyme reactors?

Answer: Following are the parameter affecting the performance of enzyme reactors :

I. The vessel should be capable of being operated aseptically for a number of days and should be reliable in long-term operation and meet the requirements of containment regulations.

II. Adequate aeration and agitation should be provided to meet the metabolic requirements of the micro-organism. However, the mixing should not cause damage to the organism.

III. Power consumption should be as low as possible.IV. A system of temperature control should be provided.V. A system of pH control should be provided.

VI. Sampling facilities should be provided.VII. Evaporation losses from the fermenter should not be excessive.

VIII. The vessel should be designed to require the minimal use of labour in operation, harvesting, cleaning and maintenance.

IX. Ideally the vessel should be suitable for a range of processes, but this may be restricted because of containment regulations.

X. The vessel should be constructed to ensure smooth internal surfaces, using welds instead of flange joints whenever possible.

XI. The vessel should be of similar geometry to both smaller and larger vessels in the pilot plant or plant to facilitate scale-up.

XII. The cheapest materials which enable satisfactory results to be achieved should be used.

XIII. There should be adequate service provisions for individual plantsXIV. Reactor with the enzyme immobilized in or on a membrane that may at the same

time separate two phases such as water and organic solvent. XV. reactor with physically separated enzyme and organic solvent in order to prevent

denaturation of the protein

96. Discuss the various operational strategies of immobilized enzyme reactors.

Answer: Followings are the various operational strategies of immobilized enzyme reactors:

• The final decision for a certain reactor design should be based on an optimization process covering all relevant factors contributing to the overall costs, including investment, catalyst consumption, or productivity. The decision as to specific reactor design will be based on a careful analysis of the kinetic properties of the reaction system.

• For example, if the enzyme shows a strong substrate-surplus inhibition, a continuously operated reactor with complete backmixing working at high conversion is advantageaous.

• The principles developed for general heterogeneous catalysis in synthetic chemistry are valid, resulting in well-known reactor configurations.Differences between enzyme catalysis and other systems result from the nature of the biocatalyst and reaction medium. For example, soft particles containing the biocatalyst, such as alginate beads, may limit the pressure drop in fixed-bed reactors.

• A reaction with strong product inhibition may utilize a batch reactor or a plug flow reactor to achieve higher volume and catalyst specific productivities. An extractive bioreactor may be used if substrates and products show different solubilities. The aqueous phase containing the enzyme will be saturated until the maximum

solubility of with the substrate is reached. Reactions using biocatalysts are normally performed in aqueous solution at temperatures between 10 and 80 C and at ambient pressure.

• By using this reactor configuration, the destabilizing effect of organic solvents may also be overcome, because the enzyme is separated from the organic phase, which is used to extract the insoluble product.

• Due to the inhibition of some enzymes by heavy metals, the materials of construction must not release these elements.

• Reactors are operated under conditions that prevent microbial contamination. The reactor itself as well as the substrate may be sterilized prior to reaction by using chemical agents (ethanol, formaldehyde, ethylenoxide, Velcorin) or steam. Ultraviolet rays may be used to sterilize the immobilized enzyme on its support.

• Alternatively, the immobilization may be performed under sterile conditions. Antibacterial agents may be added to the reaction mixture to prevent microbial growth while the reactor is running. In some cases, the reactants may act as sterilants or inhibitors of microbial growth, such as ketones or alcohols.

• At higher concentrations (more than 500 mmol/L), solutions may become autosterile because of osmotic pressure effects. Industrial processes are often performed at elevated temperatures, above 55 C, reducing the danger of microbial

contamination. For a constant product quality and reproducibility of downstream processing, the reactor should be operated at constant conversion.

• To overcome the deactivation per unit of time that shows all biocatalysts as a result of denaturation processes, either the residence time has to be increased or fresh enzyme has to be supplied.The latter is especially easy for soluble enzymes. For carrier-fixed enzymes, a combination of both methods is used, as discussed later.

97. What do you mean by STR and CSTR enzyme reactors?

Answer: Refer to answer of question no. 83,84 ,85

98. What are the merits and demerits of different enzyme reactors ?

Each reactor contains hundreds of such fibers into which the enzyme is retained; usually

these reactors use soluble enzymes. The substrate is kept in the main chamber of the

reactor. These reactors can also be used in a continuous mode and the substrate flow rate is

adjusted to achieve the desired level of reaction. These reactors are,

(i) easy to establish,

(ii) permit the use of more than one enzyme to catalyze a chain of reactions,

(iii) allow easy replacement of enzymes, and

(iv) are useful in producing small scale (g to kg) quantities.

The chief limitations of these systems are:

(i) regular replacement of membranes adds to cost and,

(ii) the need for substrate diffusion through the membrane often limits its applications.

99. Explain use of fluidized Bed Bioreactors (FBB)

Answer : Fluidized bed bioreactors (FBB) have received increased attention in the recent years due to their advantages over other types of reactors.· Most of the FBBs developed for biological systems involving cells as biocatalysts are three phase systems (solid, liquid & gas).· The FBBs are generally operated in co-current upflow with liquid as continuous phase and other more unusual configurations like the inverse three phase fluidized bed or gas solid fluidized bed are not of much importance.

· Usually fluidization is obtained either by external liquid recirculation or by gas fed to the reactor.· In the case of immobilized enzymes the usual situation is of two-phase systems involving solid and liquid but the use of aerobic biocatalyst necessitate introduction of gas (air) as the third phase.· A differentiation between the three phase fluidized bed and the airlift bioreactor would be made on the basis that the latter have a physical internal arrangement (draft tube), which provides aerating and non-aerating zones.· The circulatory motion of the liquid is induced due to the draft tube.· Basically the particles used in FBBs can be of three different types:i. inert core on which the biomass is created by cell attachmentii. Porous particles in which the biocatalyst is entrapped. iii. Cell aggregates/ flocs (self-immobilization).· In comparison to conventional mechanically stirred reactors, FBBs provide a much lower attrition of solid particles.· The biocatalyst concentration can significantly be higher and washout limitations of free cell systems can be overcome.· In comparison to packed bed reactors FBBs can be operated with smaller size particles without the drawbacks of clogging, high liquid pressure drop, channeling and bedcompaction. The smaller particle size facilitates higher mass transfer rates and better mixing.· The volumetric productivity attained in FBBs is usually higher than in stirred tank and packed bed bioreactors. There are several successful examples of using FBBs inbioprocess development.

100. Explain use of packed Bed Bioreactors.

Answer : · Packed bed or fixed bed bioreactors are commonly used with attached biofilms especially in wastewater engineering. · The use of packed bed reactors gained importance after the potential of whole cell immobilization technique has been demonstrated.· The immobilized biocatalyst is packed in the column and fed with nutrients either from top or from bottom.· One of the disadvantages of packed beds is the changed flow characteristic due to alterations in the bed porosity during operation.· While working with soft gels like alginates, carragenan etc the bed compaction which generally occurs during fermentation results in high pressure drop across the bed.· In many cases the bed compaction was so severe that the gel integrity was severely hampered. In addition channeling may occur due to turbulence in the bed.· Though packed beds belong to the class of plug flow reactors in which backmixing is absent in many of the packed beds slight amount of backmixing occurs whichchanges the characteristics of fermentation.

· Packed beds arc generally used where substrate inhibition governs the rate of reaction.· The packed bed reactors are widely used with immobilized cells.· Several modifications such as tapered beds to reduce the pressure drop across the length of the reactor, inclined bed, horizontal bed, rotary horizontal reactors have been tried with limited success.

Unit III

101. Q. Explain Biosynthesis of Protein. Answer: Translation is the enzymatic process in which mRNA is decoded or translated into protein with the

help of ribosome and tRNA.

Translation involves the conversion of a four base code (ATCG) into twenty different

amino acids. A codon or triplet of bases specifies a given amino acid. Most amino acids

are specified by more than one codon.

The conversion of codon information into proteins is conducted by transfer RNA. Each

transfer RNA (tRNA) has an anticodon which cans base pair with a codon on mRNA.

Some anti-codons have modified bases that can pair with more than one codon,

specifying the same amino; this means that we don't need 61 different tRNA molecules

for all 61 codons.

In translation, messenger RNA (mRNA) produced by transcription is decoded by the ribosome to

produce a specific amino acid chain, or polypeptide, that will later fold into an active protein.

Translation occurs in the cytoplasm of both prokaryotic (Pr) and eukaryotic (Eu) cells.

In prokaryotes, ribosome can begin translating the mRNA even before RNA polymerase completes

its transcription.

In eukaryotes, translation and transcription are completely separated in time and space in which

transcription occur in the nucleus and translation occur in the cytoplasm.

In Eukaryotes, translation occurs across the membrane of the endoplasmic reticulum in a process

called vectorial synthesis .

Proteins are synthesized from the amino to the carboxyl terminus.

Translation proceeds in four phases: activation, initiation, elongation and termination 

(1)Phase of Activation

The amino acids have no direct affinity for mRNA, so tRNA act as an adapter molecule, which

recognizes an amino acid on one end and its corresponding codon on the other, is required for

translation.

In activation phase, the correct amino acid is covalently bonded to the corresponding  tRNA). The

amino acid is joined by its carboxyl group to the 3' OH of the tRNA by a ester bond. When the

tRNA has an amino acid linked to it, it is termed "charged". 

As tRNAs enter the cytoplasm, each combines with its cognate amino acid in a two-step process

called amino acid activation.

Each type of amino acid is activated by a different amino acyl tRNA synthetase. Two high-energy bonds from an ATP are required. . The aminoacyl tRNA synthetase transfers the activated amino acid to the 3' end of the correct

tRNA. . The amino acid is linked to its cognate tRNA with an energy-rich bond. This bond will later supply energy to make a peptide bond linking the amino acid into a protein. Aminoacyl tRNA synthetases have self-checking functions to prevent incorrectly paired amino acyl tRNAs from forming.

(2)Phase of Initiation Initiation occurs by binding of the 30s subunit to the mRNA. In prokaryotes, the 165 rRNA of the small subunit binds to the Shine-Dalgarno sequence in the 5'

untranslated region of the mRNA. In eukaryotes, the small subunit binds to the 5' cap structure and slides down the message to the

first AUG. The charged initiator tRNA becomes bound to the AUG start codon on the message through base

pairing with its anticodon. The initiator tRNA in prokaryotes carries fmet, whereas the initiator tRNA in eukaryotes carries

Met. The large subunit binds to the small subunit, forming the completed initiation complex. There are two important binding sites on the ribosome called the P site and the A site.

o The peptidyl site (P site) is the site on the ribosome where (f)met-tRNA i initially binds. After formation of the first peptide bond, the P site is a binding site for the growing peptide chain.

o The aminoacyl site (A site) binds each new incoming tRNA molecule carrying an activated amino acid.

(3)Phase of Elongation

Elongation occurs by successive amidation of the nascent (growing) chain. Elongation is a three-step cycle that is repeated for each amino acid added to the protein after the

initiator methionine. Each cycle uses four high-energy bonds in which two from the ATP used in amino acid activation

to charge the tRNA, and two from GTP. During elongation, the ribosome moves in the 5' to 3' direction along the mRNA, synthesizing the

polypeptide chain from amino to carboxyl terminus. The three steps are:

o A charged tRNA binds in the A site. The particular aminoacyl-tRNA is determined by the mRNA codon aligned with the A site.

o Peptidyl transferase, an enzyme that is part of the large subunit, forms the peptide bond between the new amino acid and the carboxyl end of the growing polypeptide chain. The bond linking the growing peptide to the tRNA in the P site is broken, and the growing peptide attaches to the tRNA located in the A site.

o In the translocation step, the ribosome moves exactly three nucleotides (one codon) along the message. This moves the growing peptidyl-tRNA into the P site and aligns the next codon to be translated with the empty A site.

In eukaryotic cells, elongation factor-2 (eEF-2) used in translocation is inactivated throughADP-ribosylation by Pseudomonas and Diphtheria toxins.

(4)Phase of Termination

Termination occurs any of the three stop (termination or nonsense) codons moves into the A site. This process is facilitated by a releasing factor protein that binds into the ribosomal A site containing a stop codon.

With the help of release factor peptidyl transferase hydrolyzes the completed protein from the final tRNA in the P site.

Since no tRNA exists with an anticodon complementary to the stop codon, the ribosome "pauses" until at last it "falls off" the mRNA, and the polypeptide chain terminates.

The mRNA, ribosome, tRNA, and factors can all be reused for additional protein synthesis.

Fig: Steps in Translation

Pr = Prokaryote Eu= Eukaryote

102. Q. Explain effect of amino acids on structure of proteinsAnswer: Amino acids are the building blocks of proteins. An alfa-amino acid consistsof a central carbon atom, called the alfa carbon, linked to an amino group, acarboxylic acid group, a hydrogen atom, and a distinctive R group. The R group is often referred to as the side chain. With four different groups connected to the tetrahedral _-carbon atom, _-amino acids are chiral; the two mirror-image forms are called the L isomer and the D isomer. Only L amino acids are constituents of proteins

The polypeptide chain folds up to form a specific shape (conformation) inthe protein. This conformation is the three-dimensional arrangement of atomsin the structure and is determined by the amino acid sequence.

The bulky and relatively inflexible Pro and Hyp residues confer rigidity on the entire assembly. Valine is helix destabilizing .Glycine present on sharp turn position.

The non-polar residues Val, Leu, Ile, Met, and Phe occur mostly in the interior of a protein, out of contact with the aqueous solvent. The hydrophobic effects that promote this distribution are largely responsible for the three-dimensional structure of native proteins.

The charged polar residues Arg, His, Lys, Asp, and Glu are usually located on the surface of a protein in contact with the aqueous solvent. This is because immersing an ion in the virtually anhydrous interior of a protein is energetically unfavorable.

The uncharged polar groups Ser, Thr, Asn, Gln, and Tyr are usually on the protein surface but also occur in the interior of the molecule. When buried in the protein, these residues are almost always hydrogen bonded to other groups; in a sense, the formation of a hydrogen bond “neutralizes” their polarity. This is also the case with the polypeptide backbone.

These general principles of side chain distribution are evident in individualelements of secondary structure as well as in whole proteins Polar side chains tend to extend toward—and thereby help form—the protein’s surface, whereas nonpolar side chains largely extend toward—and thereby occupy—its interior.

Most proteins are quite compact, with their interior atoms packed togethereven more efficiently than the atoms in a crystal of small organic molecules. Nevertheless, the atoms of protein side chains almost invariably have low-energy arrangements. Evidently, interior side chains adopt relaxed conformations despite the profusion of intramolecular interactions.

Closely packed protein interiors generally exclude water. When water molecules are present, they often occupy specific positions where they can form hydrogen bonds, sometimes acting as a bridge between wo hydrogen-bonding protein groups.

The aggregation of nonpolar side chains in the interior of a protein is favored by the increase in entropy of the water molecules that would otherwise form ordered “cages” around the hydrophobic groups.The combined hydrophobic and hydrophilic tendencies of individual aminoacid residues in proteins can be expressed as hydropathies. The greater a side chain’s hydropathy, the more likely it is to occupy the interior of a protein and vice versa.The association of two ionic protein groups of opposite charge (e.g., Lysand Asp) is known as an ion pair or salt bridge. About 75% of the chargedresidues in proteins are members of ion pairs that are located mostly onthe protein surface.Disulfide bonds within and between polypeptide chains form as a protein folds to its native conformation.Metal ions may also function to internally cross-link proteins. For example,at least ten motifs collectively known as zinc fingers have been described in nucleic acid–binding proteins. These structures contain about 25–60 residues arranged around one or two Zn21 ions that are tetrahedrally coordinated by the side chains of Cys, His, and occasionally Asp or Glu.Q. Briefly introduce about the conformational study of protein?Answer:

Q. What do you mean by Ramachandran plot?

Unit IV

103. Q. Explain the physical methods such as X-ray crystallography for determination of protein structure.

Answer: X-Ray crystallography is one of the most powerful methods for studying macromolecular structure. It is the primary method for determining the molecular conformations of biological macromolecules, particularly protein and nucleic acids. The first crystal structure of a macromolecule was solved in 1958. A three-dimensional model of the myoglobin molecule obtained by X-ray analysis. The Protein Data Bank (PDB) is a freely accessible repository for the structures of proteins and other biological macromolecules. Computer programs like RasMol or Pymol can be used to visualize biological molecular structures

According to optical principles, the uncertainty in locating an object is approximately equal to the wavelength of the radiation used to observe it. X-Rays can directly image a molecule because X-ray wavelengths are comparable to covalent bond distances (~1.5 Å) An individual molecules cannot be seen in a light microscope because visible light has a minimum wavelength of 4000 Å.

When a crystal of the molecule to be visualized is exposed to a parallel beam of X-rays, the atoms in the molecule scatter the X-rays, with the scattered rays canceling or reinforcing each other in a process known as diffraction. The resulting diffraction pattern is recorded on photographic film (Fig.1) or by a radiation counter. The intensities of the diffraction maxima (darkness of the spots on the film) are then used to mathematically construct the three-dimensional image of the crystal structure.

An intuitive understanding of X-ray diffraction can be obtained from the Bragg model of diffraction. In this model, a given reflection is associated with a set of evenly spaced sheets running through the crystal, usually passing through the centers of the atoms of the crystal lattice. The orientation of a particular set of sheets is identified by its three Miller indices (h, k, l), and let their spacing be noted by Bragg which proposed a model in which the incoming X-rays are scattered mirror-like from each plane; from that assumption, X-rays scattered from adjacent planes will combine constructively (constructive interference) when

the angle θ between the plane and the X-ray results in a path-length difference that is an integer multiple n of the X-ray wavelength λ.

Where n = number of plane in the protein crystal d = distance between the adjacent plane of the cristal λ = wavelength of the X – ray

A reflection is said to be indexed when its Miller indices (or, more correctly, its reciprocal lattice vector components) have been identified from the known wavelength and the scattering angle 2θ.

Fig 1: An X-ray diffraction photograph of a crystal of sperm whale myoglobin. The intensity of each diffraction maximum (the darkness of each spot) is a function of the crystal’s electron density.

The photograph in Fig 1 represents only a small portion of the total diffraction information available from a crystal of myoglobin, a small globular protein. In contrast, fibrous proteins do not crystallize but, instead, can be drawn into fibers whose X-ray diffraction patterns contain only a few spots and thus contain comparatively little structural information. Similarly the diffraction pattern of a DNA fiber is relatively simple.

X-Rays interact almost exclusively with the electrons in matter, not with the atomic nuclei. An X-ray structure is therefore an image of the electron densityof the object under study. This information can be shown as a three dimensionalcontour map (Fig. 3).

Fig 3 An electron density map. The three-dimensional outline of the electron density (orange) is shown with a superimposed atomic model of the corresponding polypeptide segment (white). This structure is a portion of human rhinovirus

Hydrogen atoms, which have only one electron, are not visible in macromolecular X-ray structures. The X-ray structures of small organic molecules can be determined with a resolution on the order of ,1 Å. Few protein crystals have this degree of organization. Furthermore, not all proteins can be coaxed to crystallize,that is, to precipitate in ordered three-dimensional arrays.

The protein crystals in Fig. 4 differ from those of most small organic molecules in being highly hydrated; protein crystals are typically 40 to 60% water by volume. The large solvent content gives protein crystals a soft, jellylike consistency so that the molecules are typically disordered by a few angstroms. This limits their resolution to about 2 to 3.5 Å, although a few protein crystals are better ordered (have higher resolution).

Figure 4. Protein crystals. (a) Azurin from Pseudomonas aeruginosa, (b) flavodoxin from Desulfovibrio vulgaris, (c) rubredoxin from Clostridium pasteurianum, (d) azidomet myohemerythrin from the marine worm Siphonosoma funafuti, (e) lamprey hemoglobin, and ( f ) bacteriochlorophyll a protein from Prosthecochloris aestuarii. These crystals are colored because the proteins contain light-absorbing groups; proteins are colorless in the absence of such groups.

A resolution of a few angstroms is too coarse to clearly reveal the positionsof individual atoms, but the distinctive shape of the polypeptide backbonecan usually be traced. The positions and orientations of its side chains can therefore be deduced. However, since many side chains have similar sizes and shapes, knowledge of the protein’s primary structure is required to fit the sequence of amino acids to its electron density map. Mathematical techniques can then refine the atomic positions to within ,0.1 Å in higher solution structures.

Another consequence of the large solvent content of protein crystals is that crystalline proteins maintain their native conformations and therefore their functions. Indeed, the degree of hydration of proteins in crystals is similar to that in cells. Thus, the X-ray crystal structures of proteins often provide a basis for understanding their biological activities. Crystal structures have been used as starting points for designing drugs that can interact specifically with target proteins under physiological conditions.

Recent advances in NMR spectroscopy have permitted the determination of the structures of proteins and nucleic acids in solution (but limited to a size of ,30 kD). Thus, NMR techniques can be used to elucidate the structures of proteins and other macromolecules that fail to crystallize.In the several cases in which both the X-ray and NMR structures of a particular protein were determined, and the two structures had few, if any, significant differences.

104 . Q. What do you mean by SubtilisinAnswer:

105 . Q. Write short notes on Tryesyl t RNA synthetase,Answer:

106 Q What do you mean by Site directed mutagenesis

107. Q .Explain basic concept for designing a new protein/enzyme molecule.

Basic concept for designing a new protein/enzyme molecule

As we know that large number of proteins/enzymes found in nature, which fold into a variety of different structures and carry out a huge diversity of functions. Protein engineering attempts to design protein/enzyme structures, including those having particular functions. Therefore targets for designing a enzyme/ protein are

I. enhancement of enzyme activity,II. improved stability of the protein,

III. altered pH optima or temperature tolerance and IV. modified specificity

Researchers can improve the knowledge we have about the forces and effects that specify

the properties of the folded states of a protein. In addition, control over the design of

particular folded state structures will likely lead to new synthetic proteins having the

efficiency and specificity of biological proteins.

Designing a new protein/enzyme molecule has applications include therapeutics, sensors,

catalysts, and materials. The successful design of proteins/enzymes is possible even without

a complete quantitative understanding of all the forces involved in specifying their

structures.

Designing of proteins/enzymes is nontrivial, however, because of both their complexity and

the delicacy of the interactions that specify the folded state. Proteins are macrobiomolecule

and having many structural variables specify the folded state, including sequence, backbone

topology, and side-chain conformations.

There are two main motivations for designing of proteins/enzymes:

The first is based upon the assumption that a complete understanding of any

natural system depend upon our ability to design a similar artificial system from first

principles.

The second motivation is for de novo protein design is one of the practical

approaches. Therefore our understanding of natural proteins for their folding

pathways, thermodynamic stabilities and catalytic properties is enhanced by our

ability to design novel proteins with predetermined structure and properties. The

ability to design proteins/enzymes de novo has the potential to bring a revolution in

the field of science and technology ranging from industrial catalyst to biomedical

engineering.

Protein/enzyme design also refers to the effort to design new protein molecules of a desired

3-D structure and function. It is a reverse procedure of protein structure prediction and the

solution of the problem therefore highly relies on the extent of our understanding on the

principle of protein folding.

Finding out an amino acid sequence that will adopt a unique and stable three dimensional

structure is the main goal to design a novel protein. The fundamental hurdle to design a

novel protein is the conformational entropy of the linear polymer chain which must be

overcome. The conformational entropy represents a substantial amount of unfavorable free

energy. For a design to succeed the favorable free energy associated with these designed

interactions must outweigh the entropic cost of fixing the chain into a unique structure.