Unit-I: Enzymes, Coenzymes and catalysis (Paper-I)

1. Thermodynamics of catalysis, Energy of activation, Relation of ∆G and Keq

2. Coupled reactions (endergonic and exergonic) in Biochemical pathways

3. Methods to isolate and purify enzymes, Assays, Activity Units, Specific activity

4. Nomenclature and classification of enzymes: EC, SCOP and CATH

5. Metal, co-factor and co-enzyme requirements

6. Vitamin cofactors: TPP, FMN/FAD, NAD/NADP, Pantothenic acid

7. Vitamin cofactors: PLP, Biotin, Folate, Cobalamine, Phylloquinonecals to identify active sites residues:

8. Factors affecting catalysis (pH, temperature, pressure, enzyme and substrate concentration)

9. Chemicals to identify active sites residues: Arg, Cys, Lys, His

10. Site- directed mutagenesis to identify active site residues: Triose Phosphate Isomerase

Unit-III: Bacterial Genetics (Paper-III)

1. Discovery of conjugation

2. Mapping bacterial genes by conjugation

3. Discovery of transformation

4. Mapping bacterial genes by transformation

5. Discovery of transduction

6. Mapping bacterial genes by transduction

7. Discovery of transposition

8. Structure of transposons, replicative and conservative transposition use as mutagenes

9. Mapping phage genes – Fine structure of rII locous: Complementation analysis

10. Fine structure of rII locus: Deletion mapping

Unit-IV: Protein Sorting, Targeting and degradation (Paper-II)

1. Post translational modifications of proteins role in targeting (isoprenylation)

2. Signal peptide (ERLS), role of SRP in translation of secreted proteins

3. NLS, Mitochondrial and chloroplast LS

4. Chaperones, HSPs in protein folding

5. Lysosomal pathways (endocytosis, crinophagy, macroautophagy, microauotophagy, direct translocation from cytosol)

6. Lysosomal storage diseases

7. Ubiquitin-proteasome pathway, N-end rule

8. Immune-proteasomes Misfolded proteins in neurodegenerative diseases

9. PEST sequence and proteolysis

10. Action of cytotoxic, hemotoxic, myotoxic and hemorrhagic venoms

History

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Enzyme Classification

The 1st 3-D structure for an enzyme was reported in 1965!

Since then a lot of progress has been made. The chemical

reaction mechanisms for enzymes and the functions of amino

acids in the active sites of several enzymes are now known.

Older systems used the following system:

Alcohol dehydrogenase

Substrate Catalytic action -ase suffix

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EC Nomenclature

Enzymes are classified according the report of a Nomenclature Committee appointed by the International Union of Biochemistry (1984).

This enzyme commission assigned each enzyme a recommended name and a 4-part distinguishing number.

However, some alternative names remain in common usage.

The enzyme commission (EC) numbers divide enzymes into six main groups according to the type of reaction catalysed:

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1. Oxidoreductases

Oxidoreductases involve redox reactions in which hydrogen or oxygen atoms or electrons are transferred between molecules.

This extensive class includes

dehydrogenases (hydride transfer),

oxidases (electron transfer to molecular oxygen),

oxygenases (oxygen transfer from molecular oxygen), and

peroxidases (electron transfer to peroxide).

Example: glucose oxidase (EC 1.1.3.4, systematic name, b-D-glucose:oxygen 1-oxidoreductase).

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1. Oxidoreductases

Oxidoreductases involve redox reactions in which hydrogen or oxygen atoms or electrons are transferred between molecules.

Example: glucose oxidase (EC 1.1.3.4, systematic name, b-D-glucose:oxygen 1-oxidoreductase).

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2. Transferases

Transferases catalyse the transfer of an atom or group of atoms (e.g. acyl-, alkyl- and glycosyl-) between two molecules.

For example: aspartate aminotransferase (EC 2.6.1.1, systematic name, L-aspartate:2-oxoglutarate aminotransferase; also called glutamic-oxaloacetic transaminase or simply GOT)

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3. Hydrolases

Hydrolases involve hydrolytic reactions and their reversal.

This is presently the most commonly used class of enzymes in

enzyme technology

includes esterases, glycosidases, lipases and proteases.

For example: chymosin (EC 3.4.23.4, no systematic name

declared; also called rennin)

k-casein + water para-k-casein + caseino macropeptide

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4. Lyases• Lyases involve elimination reactions in which a group of atoms is

removed from the substrate.

• This includes aldolases, decarboxylases, dehydratases and some

pectinases but does not include hydrolases.

• For example: histidine ammonia-lyase (EC 4.3.1.3, systematic

name, L-histidine ammonia-lyase; also called histidase)..

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5. Isomerases

• Isomerases catalyse molecular isomerisations and includes the

epimerases, racemases and intramolecular transferases.

• For example: xylose isomerase (EC 5.3.1.5, systematic name,

D-xylose ketol-isomerase; commonly called glucose isomerase).

a-D-glucopyranose a-D-fructofuranose

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6. Ligases Ligases, also known as synthetases, catalyze formation of a

covalent bond joining two molecules together, coupled with the hydrolysis of a nucleoside triphosphate.

They form a relatively small group of enzymes

For example: glutathione synthase (EC 6.3.2.3, systematic name, g-L-glutamyl-L-cysteine:glycine ligase (ADP-forming); also called glutathione synthetase).

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6. Ligases Ligases catalyze formation of a covalent bond joining two

molecules together, coupled with the hydrolysis of a NTP

For example: glutathione synthase (EC 6.3.2.3, systematic name, g-L-glutamyl-L-cysteine:glycine ligase (ADP-forming); also called glutathione synthetase).

ATP + γ-L-glutamyl-L-cysteine + glycine ADP + phosphate + glutathione

Every enzyme code consists of the letters "EC" followed by four numbers separated by periods. Those

numbers represent a progressively finer classification of the enzyme.

For example, the tripeptide aminopeptidases have the code "EC 3.4.11.4", whose components indicate the

following groups of enzymes:

•EC 3 enzymes are hydrolases (enzymes that use water to break up some other molecule)

•EC 3.4 are hydrolases that act on peptide bonds

•EC 3.4.11 are those hydrolases that cleave off the amino-terminal amino acid from a polypeptide

•EC 3.4.11.4 are those that cleave off the amino-terminal end from a tripeptide

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Formation Of ES Complex

Two models:

Lock and Key

Induced Fit

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Lock And Key Model Enzymes are very specific for substrates Emil Fischer suggested in 1894 that enzyme and the substrate possess

specific complementary geometric shapes that fit exactly into one another. They combine to form a short-lived enzyme-substrate complex.

This is often referred to as "the lock and key" model. This model explains enzyme specificity It does not explain how transition state is stabilized.

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Induced Fit Model Modification of "lock and key" model in 1958 by Daniel Koshland Enzymes are flexible. In this model, E changes shape as the S molecules bind - the change in

shape is 'induced' by the approaching S molecules. Hence, active site of E can be modified upon interaction with S. In some cases, S changes shape (slightly) upon binding E. Unlike the "Lock and key" model, this model explains specificity and

stabilization of the transition state.

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Factors Affecting Rate: [E]

The rate of enzyme-catalyzed reaction increases with concentration of enzyme and substrate.

Rate increases linearly with [E] if [S] is not limiting.

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Factors Affecting Rate: [S]

At a fixed [E], rate increases exponentially with increasing [S] up to a point.

After that, rate does not increase significantly with increase [S].

This is because the E (active site) are virtually saturated with substrate.

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Factors Affecting E: Temperature

KE of molecules increases with T

Increased KE increases chances of a successful collision

Thus rate increases with T

This rise is seen only till a certain temperature.

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Factors Affecting E: Temperature

The temperature at which an enzyme's catalytic activity is at its greatest = optimal temperature (~37.5ºC for enzymes in human cells)

Above this temperature enzyme structure begins to denature - at higher temperatures intra- and inter-molecular bonds are broken as E molecules also gain kinetic energy.

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Factors Affecting E: pH

Each enzyme works within quite a small pH range.

The pH at which its activity is greatest = optimal pH.

This is because changes in pH can make and break intra- and intermolecular bonds, changing the structure of E and, therefore, its activity.

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Factors Affecting Rate: [I]

The rate of enzyme-catalyzed reaction is affected by presence of other substances that compete or interfere with the formation of ES complex.

Such substances are called inhibitors of enzyme action.

Inhibitors can bind to

Active site of E

Some other site of E

Co-factors

• Some enzymes can catalyze reactions in absence of any other factors –the protein itself is sufficient for catalysis.

• Some enzyme require an additional chemical component called a co-factor for catalytic activity.

• The co-factors are usually one or more inorganic ions, such as Fe2+, Mg2+, Mn2+, or Zn2+.

Examples of metal co-factors

• Cu2+ Cytochrome oxidase• Fe2+ or Fe3+ Cytochrome oxidase, catalase,

peroxidase• K+ Pyruvate kinase• Mg2+ Hexokinase, glucose 6-phosphatase,

pyruvate kinase• Mn2+ Arginase, ribonucleotide reductase• Mo Dinitrogenase• Ni2+ Urease• Se Glutathione peroxidase• Zn2+ Carbonic anhydrase,

alcohol dehydrogenase, carboxypeptidases A and B

Co-enzymes

• Some enzymes require an additional complex organic or metalloorganic molecule called a co-enzyme for catalytic activity.

• Co-enzymes act as transient carriers of specific functional groups. • Most co-enzymes are derived from vitamins.• A coenzyme or metal ion that is very tightly or even covalently bound to the

enzyme protein is called a prosthetic group. • A complete, catalytically active enzyme together with its bound coenzyme

and/or metal ions is called a holoenzyme.• The protein part of such an enzyme is called the apoenzyme or apoprotein.

Examples of co-enzymes

• Coenzyme Chemical groups transferred

• Biocytin CO2

• Coenzyme A Acyl groups • 5-Deoxyadenosylcobalamin H atoms and alkyl groups • FAD Electrons • Lipoate Electrons and acyl groups • NAD Hydride ion (:H) • Pyridoxal phosphate Amino groups • Tetrahydrofolate One-carbon groups• Thiamine pyrophosphate Aldehydes

Biotin

• Biotin plays a key role in many carboxylation reactions. • It is a specialized carrier of one-carbon groups in their most oxidized

form: CO2. • The transfer of one-carbon groups in more reduced forms is mediated by

other cofactors, notably tetrahydrofolate and S-adenosylmethionine.• Carboxyl groups are activated in a reaction that splits ATP and joins CO2

to enzyme-bound biotin. • This “activated” CO2 is then passed to an acceptor in a carboxylation

reaction.

Mechanism of pyruvate carboxylase

• Pyruvate carboxylase has four identical subunits.• Each subunit contains a molecule of biotin covalently

attached through an amide linkage to the ε-amino group of a specific Lys residue in the enzyme active site.

• Carboxylation of pyruvate proceeds in two steps.: 1. A carboxyl group derived from HCO3

- is attached to biotin.2. The carboxyl group is transferred to pyruvate to form

oxaloacetate. • These two steps occur at separate active sites; the long

flexible arm of biotin transfers activated carboxyl groups from the first active site to the second.

Pyruvate carboxylase

1. Bicarbonate is activated by ATP to form carboxyphosphate 2. Carboxyphosphate breaks down to carbon dioxide

Pyruvate carboxylase

3. Carbon dioxide reacts with biotin to form carboxy-biotin.

Pyruvate carboxylase4. CO2 is transferred to

2nd catalytic site.

5 -7. CO2 reacts with pyruvate enolate to form oxaloacetate.

Pyruvate carboxylase

1. Bicarbonate is activated by ATP to form carboxyphosphate 2. Carboxyphosphate breaks down to carbon dioxide

Pyruvate carboxylase

1. Bicarbonate is activated by ATP to form carboxyphosphate 2. Carboxyphosphate breaks down to carbon dioxide

“Long arms”

• The co-factors lipoate, biotin, and the combination of -mercaptoethylamine and pantothenate can bind covalently to enzymes to form long, flexible arms.

• These arms act as tethers that move intermediates from one active site to the next.

• The group shaded pink is the point of attachment of the activated intermediate to the tether.

Co-enzyme B12

• Coenzyme B12 is the cofactor form of vitamin B12,• It is unique among all the vitamins in that it contains not only a complex organic

molecule but an essential trace element, cobalt. • The complex corrin ring system of vitamin B12 to which cobalt (as Co3) is coordinated,

is chemically related to the porphyrin ring system of heme and heme proteins. • A fifth coordination position of cobalt is filled by dimethylbenzimidazole

ribonucleotide, bound covalently by its 3-phosphate group to a side chain of the corrin ring, through aminoisopropanol.

• The formation of this complex cofactor occurs in one of only two known reactions in which triphosphate is cleaved from ATP; the other reaction is the formation of S-adenosylmethionine from ATP and methionine.

Co-enzyme B12

• The 3D structure of the cofactor was determined by Dorothy Crowfoot Hodgkin in 1956.

• Vitamin B12 as usually isolated is called cyanocobalamin, because it contains a cyano group (picked up during purification) attached to cobalt in the 6th coordination position.

• In 5-deoxyadenosylcobalamin, the cyano group is replaced by the 5-deoxyadenosyl group covalently bound through C-5 to the cobalt.

Mechanism

1. E breaks the CoOC bond in the cofactor, leaving the coenzyme in its Co2+ form and producing a 5-deoxyadenosyl free radical.

2. This radical abstracts a H-atom from the substrate, converting the substrate to a radical and producing 5-deoxyadenosine.

Mechanism

3. This radical yields another radical, in which the migrating group X (OCOOS-CoA for methylmalonyl- CoA mutase) has moved to the adjacent carbon to forma radical that has the carbon skeleton of the eventual product (a four-carbon straight chain). The H-atom initially abstracted from the substrate is now part of the OCH3 group of 5-deoxyadenosine.

4. One of the H from this same OCH3 group is returned to the productlike radical, generating the product and regenerating the deoxyadenosyl free radical.

Mechanism

5. The bond re-forms between cobalt and the OCH2 group of the deoxyadenosyl radical, destroying the free radical and regenerating the cofactor in its Co3 form, ready to undergo another catalytic cycle.

In this postulated mechanism, the migrating hydrogen atom never exists as a free species and is thus never free to exchange with the hydrogen of surrounding water molecules.

Mechanism• The key to understanding

how coenzyme B12 catalyzes H- exchange lies in the properties of the covalent bond between cobalt and C-5 of the deoxyadenosyl group.

• This is a relatively weak bond; its bond dissociation energy is ~ 110 kJ/mol, compared with 348 kJ/mol for a typical COC bond or 414 kJ/mol for a COH bond.

• This bond is highly photo-labile (possible reason for the absence of vitamin B12 in plants).

• Dissociation produces a 5-deoxyadenosyl radical and the Co2+.

Pyridoxal Phosphate

• All aminotransferases have the same prosthetic group and the same reaction mechanism.

• The prosthetic group is pyridoxal phosphate (PLP), the coenzyme form of pyridoxine, or vitamin B6.

• Pyridoxal phosphate functions as an intermediate carrier of amino groups at the active site of aminotransferases.

• It undergoes reversible transformations between its aldehyde form, pyridoxal phosphate, which can accept an amino group, and its aminated form, pyridoxamine phosphate, which can donate its amino group to an -keto acid.

• Pyridoxal phosphate is generally covalently bound to the enzyme’s active site through an aldimine (Schiff base) linkage to the –amino group of a Lys residue.

PLP• Aminotransferases are classic examples of enzymes catalyzing bimolecular Ping-Pong reactions, in which the first substrate reacts and the product must leave the active site before the second substrate can bind.

• Thus the incoming amino acid binds to the active site, donates its amino group to pyridoxal phosphate, and departs in the form of an –keto acid.

• The incoming -keto acid then binds, accepts the amino group from pyridoxamine phosphate, and departs in the form of an amino acid.

PLP

• Pyridoxal phosphate (PLP) and its aminated form, pyridoxamine phosphate, are the tightly bound coenzymes of aminotransferases.

• Pyridoxal phosphate is bound to the enzyme through noncovalent interactions and a Schiffbase linkage to a Lys residue at the active site.

PLP

• Some transformations:

Co-factors for 1-C atom transfers

• Biotin transfers carbon in its most oxidized state, CO+• Tetrahydrofolate transfers one-carbon groups in intermediate

oxidation states and sometimes as methyl groups• S-adenosylmethionine transfers methyl groups, the most

reduced state of carbon.

Tetrahydrofolate

• Tetrahydrofolate (H4 folate), synthesized in bacteria, consists of substituted pterin (6-methylpterin), p-aminobenzoate, and glutamate moieties.

• The oxidized form, folate, is a vitamin for mammals; it is converted in two steps to tetrahydrofolate by the enzyme dihydrofolate reductase.

• The one-carbon group undergoing transfer, in any of three oxidation states, is bonded to N-5 or N-10 or both.

• The most reduced form of the cofactor carries a methyl group, a more oxidized form carries a methylene group, and the most oxidized forms carry a methenyl, formyl, or formimino group.

• The primary source of one-carbon units for tetrahydrofolate is the carbon removed in the conversion of serine to glycine, producing N5,N10-methylenetetrahydrofolate.

The different molecular species are grouped according to oxidation state, with the most reduced at the top and most oxidized at the bottom.

All species within a single shaded box are at the same oxidation state. The conversion of N5,N10-methylenetetrahydrofolate to N5-methyltetrahydrofolate is effectively irreversible.

Tetrahydrofolate

• The enzymatic transfer of formyl groups, as in purine synthesis and in the formation of formylmethionine in prokaryotes, generally uses N10-formyltetrahydrofolate rather than N5-formyltetrahydrofolate.

• The latter species is significantly more stable and therefore a weaker donor of formyl groups.

• N5-formyltetrahydrofolate is a minor byproduct of the cyclohydrolase reaction, and can also form spontancously.

• Conversion of N5-formyltetrahydrofolate to N5, N10-methenyltetrahydrofolate, requires ATP, because of an otherwise unfavorable equilibrium.