enzyme

ENZYMEDr. Deepak K Gupta

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Syllabus

• Definition, classification, specificity and active site.

• Cofactors.

• Effect of pH temperature and substrate concentration.

• Introduction to enzyme inhibitors, proenzymes and isoenzymes.

• Introduction to allosteric regulation, covalent modification and regulation by induction / repression


Introduction

• Enzymes are biological catalysts synthesized by living cells that accelerate biochemical reactions.

• The orderly course of metabolic processes is only possible because each cell is equipped with its own genetically determined set of enzymes

• It is only this that allows coordinated sequencesof reactions - metabolic pathways

• Involved in many regulatory mechanisms.

• Almost all enzymes are proteins except catalytically active ribonucleic acids, the ribozymes


Enzyme• Enzymes are characterized by three distinctive

features:• Catalytic Power

– Ability to catalyses biochemical reaction– Accelerating reaction rates as much as 1016 over

uncatalyzed levels - far greater than any synthetic catalysts

• Specificity– A given enzyme is very selective– Both in the substances with which it interacts and in the

reaction that it catalyzes

• Regulation– Metabolic inhibitors and activators


Enzyme Nomenclature• Traditionally, enzymes often were named by

adding the suffix –ase to the substrate upon which they acted

• Ex: phosphatase, urease, catalase, proteases

• Confusion arose from these trivial naming.

• So a new system of nomenclature of enzyme was developed based on nature of reaction it helps

• Six classes of reactions are recognized– Within each class are subclasses, and under each

subclass are subsubclasses within which individual enzymes are listed


Classification of Enzyme

• Enzyme are classified on the basis of action it performs– Oxidoreductases - oxidation–reduction reactions

• Phosphate dehydrogenase

– Transferases - transfer of functional groups• Methyltransferases, Carboxyltransferases

– Hydrolases - hydrolysis reactions• Carboxylic ester hydrolases

– Isomerases - isomerization reactions• Epimerases

– Lyases - addition to double bonds• Carboxy lyases, Aldehyde lyases

– Ligases - formation of bonds with ATP cleavage• Amino acid–RNA ligases


Intracellular and extracellular enzymes

o Intracellular

o enzymes are synthesized and retained in the cell for the use of cell itself.

o They are found in the cytoplasm, nucleus, mitochondria and chloroplast.

Example: Oxydoreductase catalyses biological oxidation, Enzymes involved in reduction in the mitochondria.

o Extracellular

o enzymes are synthesized in the cell but secreted from the cell to work externally.

Example : Digestive enzyme produced by the pancreas, are not used by the cells in the pancreas but are transported to the duodenum. www.facebook.com/notesdental

Chemical Properties

• Most of enzymes carry out their functions relying solely on their protein structure

• Many others require non-protein components –cofactors– Usually metal ion or non-protein organic part (Coenzyme)

• Less complex than proteins, tend to be stable to heat

• Many coenzymes are vitamins or contain vitamins as part of their structure

• Functional unit of enzyme is known as holoenzyme– Holenzyme = Apoenzyme + Coenzyme

• Apoenzyme : protein without any catalytic activity


Chemical Properties

• If the enzyme is made of single polypeptide –monomeric enzyme. Ex: ribonuclease, trypsin

• If the enzyme is made up of more than one polypeptide – oligomeric enzyme. Ex: lactate dehydrogenase, aspartate transcarbamoylase

• Multienzyme complex: have multiple enzyme unit to carry out different reaction in sequence


Enzyme Kinetics

• The quantitative measurement of the rates of enzyme-catalyzed reactions and the systematic study of factors that affect these rates

• Helps in analysis, diagnosis, and treatment of the enzymic imbalances that underlie numerous human diseases.

• Levels of particular enzymes serve as clinical indicators for pathologies– myocardial infarctions,– prostate cancer– damage to the liver


Enzyme Kinetics

• Any biochemical reaction constitute,

A + B C + D

Where A and B are substrate and C + D are product

• Study of enzyme kinetic has 2 component, i.e.

• Gibs Free Energy : Direction and equilibrium state of substrate and product

• Activation Energy: Mechanism of reaction and rate of reaction


Gibs Free Energy Change ΔG

• Also called either free energy or Gibbs energy

• It describes in quantitative form both the direction in which a chemical reaction will tend to proceed and the concentrations of substrate and products that will be present at equilibrium

• Mathematically, ΔG = ΔGp – ΔGs

– ΔGp : sum of the free energies of formation of the reaction products

– ΔGs : sum of the free energies of formation of the substrates

• The sign and the magnitude of the free energy change determine how far the reaction will proceed

• If ΔG is negative then the reaction proceeds in forward direction spontaneously


Activation Energy

• Any reaction doesn’t proceeds directly to product formation.

• There is always a transition state between ground state and products

• Activation energy: The difference between the energy levels of the ground state and the transition state.

• The function of a catalyst is to increase the rate of a reaction, it does not affect reaction equilibria.

• So enzyme just lowers the activation energy.


Enzyme Kinetics


Factors effecting enzyme activity

• The contact between enzyme and substrate is the most essential pre-requisite for enzyme activity.

• The important factors that influence the enzyme reaction are– Concentration of Substrate– Concentration of Enzyme– Temperature– pH– Product concentration– Activators– Time– Light and radiation


Concentration of Substrate

• The frequency with which molecules collide is directly proportionate to their concentrations

Rate ∝ [A]n[B]m , Rate = k[A]n[B]m

– where, nA + mB → P; k = rate constant• The sum of the molar ratios of the reactants defines

the kinetic order of the reaction

• In the example above, reaction is said to be of (n+m) order overall but n order with respect to A and m order with respect to B


Michaelis-Menten Constant Km

• Also known as Haldane’s Constant

• Substrate concentration to produce half maximum velocity in an enzyme catalyst reaction

• Km is constant and a chracterstic feature of a given enzyme – strength of Enzyme Substrate (ES) complex

• Low Km value indicates a strong affinity between enzyme and substrate

• Majority of Enzyme Km value – 10-5 to 10-2


Michaelis-Menten Constant Km

Michaelis-Menten Reaction


Concentration of Enzyme

• Reaction velocity is directly proportional to concentration of enzyme

• Serum enzyme for diagnosis of disease

– Known volume of serum and substrate taken at optimum pH and temperature

– Enzyme is assayed in laboratory


Temperature

• Velocity of an enzyme reaction increase with the increase in temperature up to a maximum and then declines

• Increase in temperature causes increases the kinetic energy of molecules

• A bell-shaped curve is usually observed

• Temperature coefficient Q10 : increase in enzyme velocity when the temperature is increased by 100C

• Optimum temperature for most of enzyme – 40 – 45 0C

• Beyond 500C there is denaturation of enzyme


pH

• Most intracellular enzymes exhibit optimal activity at pH values between 6 - 8.

• Balance between enzyme denaturation at high or low pH and effects on the charged state of the enzyme, the substrates, or both

• Exception – pepsin (1-2), acid phosphatase (4-5), alkaline phophatase (10-11)


Activators

• Certain metallica cations – Mn, Mg, Zn, Ca, Co, Cu, Na, K.

• It acts in a various ways– Combining with substrate– Formation of E-S metal complex, direct participation in

the reaction and bringing a conformational changes in enzyme

• There are 2 categories of enzyme requiring metals for their activity• Metal activated enzyme:Not tightly held by the enzyme and can

be exchanged easily. Ex: ATPAase (Mg and Ca) and Enolase• Metalloenzyme: Hold the metal tightly. Ex: alcohol dehydrogenase,

carbonic anhydrase, alkaline phosphatase, carboxypeptidase


Other Factor

• Product concentration: Accumulation of reaction products generally decreases the enzyme velocity

• Light and radiation: exposure to UV, beta-gamma and X-rays inactivates certain enzyme

– Formation of peroxides, ex: UV rays inhibit salivary amylase activity


Mechanism of Enzyme Action: Active Sites

• The active site of an enzyme is the region thatbinds substrates, co-factors and prosthetic groupsand contains residue that helps to hold thesubstrate.

• Active sites generally occupy less than 5% of thetotal surface area of enzyme.

• Active site has a specific shape due to tertiarystructure of protein.

• A change in the shape of protein affects theshape of active site and function of the enzyme.


Active Sites

This model (above) is an enzyme called

Ribonuclease S, that breaks up RNA

molecules. It has three active sites (arrowed).

Active site:

The active site contains both binding

and catalytic regions. The substrate

is drawn to the enzyme’s surface and

the substrate molecule(s) are

positioned in a way to promote a

reaction: either joining two molecules

together or splitting up a larger one.Enzyme molecule:

The complexity of the

active site is what makes

each enzyme so specific

(i.e. precise in terms of the

substrate it acts on).

Substrate molecule:

Substrate molecules are the

chemicals that an enzyme

acts on. They are drawn into

the cleft of the enzyme.


Active site

o Active site can be further divided into:

it chooses the substrate It performs the catalytic

and binds it to active site. action of enzyme.

Active Site

Binding Site Catalytic Site


Mechanism of enzyme action

• The catalytic efficiency of enzymes is explained by twoperspectives:

Thermodynamic changes

Processes at the active site


Thermodynamic changes

• All chemical reactions have energy barriers between reactants andproducts.

• The difference in transitional state and substrate is called activationalbarrier.


Processes at the active site

Covalent catalysis

Acid base catalysisCatalysis

by strain

Catalysis by

proximity


Covalent catalysis

o Enzymes form covalent linkages with substrate forming transient enzyme-

substrate complex with very low activation energy.

o Enzyme is released unaltered after completion of reaction.


acid-base catalysis

• Mostly undertaken by oxidoreductases enzyme.

• Mostly at the active site, histdine is present which act as both protondonor and proton acceptor.


Catalysis by proximity

• In this catalysis molecules must come in bond forming distance.

• When enzyme binds:

A region of high substrate concentration is produced at active site.

This will orient substrate molecules especially in a position ideal for them.


Catalysis by bond strain

• Mostly undertaken by lyases.

• The enzyme-substrate binding causes reorientation of the structureof site due to in a strain condition.

• Thus transitional state is required and here bond is unstable andeventually broken.

• In this way bond between substrate is broken and converted intoproducts.


Lock and key model

• Proposed by EMIL FISCHER in 1894.

• Lock and key hypothesis assumes the active site of an enzymes are rigid inits shape.

• There is no change in the active site before and after a chemical reaction.


Lock and Key ModelThe lock and key model of enzyme action, proposed earlier this century, proposed that the substrate was simply drawn into a closely matching cleft on the enzyme molecule.

Substrate

Enzyme

Products

Symbolic representation of the lock and key model of enzyme action.

1. A substrate is drawn into the active sites of the enzyme.

2. The substrate shape must be compatible with the enzymes active site in

order to fit and be reacted upon.

3. The enzyme modifies the substrate. In this instance the substrate is

broken down, releasing two products.


Induced fit model

• More recent studies have revealed that the process is much more likely toinvolve an induced fit model(proposed by DANIAL KOSH LAND in 1958).

• According to this exposure of an enzyme to substrate cause a change inenzyme, which causes the active site to change it’s shape to allow enzymeand substrate to bind.


Induced Fit Model

More recent studies have revealed that the process is much more likely to involve an induced fit.

The enzyme or the reactants (substrate) change their shape slightly.

The reactants become bound to enzymes by weak chemical bonds.

This binding can weaken bonds within the reactants themselves, allowing the reaction to proceed more readily.

The enzyme

changes shape,

forcing the substrate

molecules to

combine.

Two substrate

molecules are

drawn into the cleft

of the enzyme.

The resulting end

product is released

by the enzyme

which returns to its

normal shape, ready

to undergo more

reactions.www.facebook.com/notesdental

39

Induced Fit Model


Changing the Active Site

• Changes to the shape of the active site will result in a loss of function. Enzymes are sensitive to various factors such as temperature & pH.

• When an enzyme has lost its characteristic 3D shape, it is said to be denatured. Some enzymes can regain their shape while in others, the changes are irreversible.


Inhibition

o The prevention of an enzyme process as a result of interaction ofinhibitors with the enzyme.

INHIBITORS:

Any substance that can diminish the velocity of an enzymecatalyzed reaction is called an inhibitor.


Types of inhibition

Inhibition

Reversible

Competitive Uncompetitive Mixed Non-competitive

Irreversible


REVERSIBLE INHIBITION

o It is an inhibition of enzyme activity in which the inhibiting molecular entity can associate and dissociate from the protein‘s binding site.

TYPES OF REVERSIBLE INHIBITION

o There are four types:

Competitive inhibition.

Uncompetitive inhibition.

Mixed inhibition.

Non-competitive inhibition.


Competitive inhibition

• In this type of inhibition, the inhibitors compete with the substrate for theactive site. Formation of E.S complex is reduced while a new E.I complex isformed.


Examples of competitive

inhibition

Statin Drug As Example Of CompetitiveInhibition:

• Statin drugs such as lipitor compete with HMG-CoA(substrate) and inhibitthe active site of HMG CoA-REDUCTASE (that bring about the catalysis of

cholesterol synthesis).


Uncompetitive Inhibition

• In this type of inhibition, inhibitor does not compete with the substrate forthe active site of enzyme instead it binds to another site known asallosteric site.


Examples of

uncompetitive

inhibition

• Drugs to treat cases of poisoning by methanol or ethylene glycolact as uncompetitive inhibitors.

• Tetramethylene sulfoxide and 3- butylthiolene 1-oxide are

uncompetitive inhibitors of liver alcohaldehydrogenase.


Non competitive

inhibition

o It is a special case of inhibition.

o In this inhibitor has the same affinity for either enzyme E or the E.Scomplex.

MIXED INHIBITION

o In this type of inhibition both E.I and E.S.I complexes are formed.

o Both complexes are catalytically inactive.


Irreversible inhibition

• This type of inhibition involves the covalent attachment of the inhibitor tothe enzyme.

• The catalytic activity of enzyme is completely lost.

• It can only be restored only by synthesizing molecules.


Examples of irreversible

inhibition

• Aspirin which targets and covalently modifies a key enzyme involved ininflammation is an irreversible inhibitor.

• SUICIDE INHIBITION :

It is an unusual type of irreversible inhibition where the enzyme convertsthe inhibitor into a reactive form in its active site.


ENZYME SPECIFICITY

• Enzymes are highly specific in nature, interacting with one or fewsubstrates and catalyzing only one type of chemical reaction.

• Substrate specificity is due to complete fitting of active site and substrate .

Example:

Oxydoreductase do not catalyze hydrolase reactions and hydrolase do notcatalyze reaction involving oxidation and reduction.


Types of enzyme

specificity

• Enzymes show different degrees of specificity:

Bond specificity.

Group specificity.

Absolute specificity.

Optical or stereo-specificity.

Dual specificity.


BOND SPECIFICITY

• In this type, enzyme acts on substrates that are similar in structure andcontain the same type of bond.

Example :

• Amylase which acts on α-1-4 glycosidic ,bond in starch dextrin andglycogen, shows bond specificity.


GROUP SPECIFICITY

• In this type of specificity, the enzyme is specific not only to the type ofbond but also to the structure surrounding it.

Example:

Pepsin is an endopeptidase enzyme, that hydrolyzes central peptide bondsin which the amino group belongs to aromatic amino acids e. g phenylalanine, tyrosine and tryptophan.


SUBSTRATE SPECIFICITY

• In this type of specificity ,the enzymes acts only on one substrate

Example :

Uricase ,which acts only on uric acid, shows substrate specificity.

Maltase , which acts only on maltose, shows substrate specificity.


OPTICAL / STEREO-SPECIFICITY

• In this type of specificity , the enzyme is not specific to substratebut also to its optical configuration

Example: D amino acid oxidase acts only on D amino acids.

L amino acid oxidase acts only on L amino acids.


DUAL SPECIFICITY

• There are two types of dual specificity.

The enzyme may act on one substrate by two different reaction types.

Example:

• Isocitrate dehydrogenase enzyme acts on isocitrate (one substrate) byoxidation followed by decarboxylation(two different reaction types) .


The enzyme may act on two substrates by one reaction type

Example:

• Xanthine oxidase enzyme acts on xanthine and hypoxanthine(twosubstrates) by oxidation (one reaction type)

DUAL SPECIFICITY


Enzyme Regulation

• Regulation of enzyme occurs in following ways

– Allosteric regulation

– Activation of Latent Enzyme

– Compartmentation

– Control of enzyme synthesis

– Enzyme Degradation

– Isoenzyme


Allosteric regulation

• Additional sites other than active sites –Allosteric enzymes

• Types of allosteric enzyme:– K-class: effectors changes the Km

– V-class: effectors changes the Vmax

• Most of allosteric enzymes are oligomeric in nature

• Non-reversible binding of effector molecule at the allosteric sites – conformational change in the active site of enzyme


Activation of Latent Enzyme

• Some enzymes remain inactive,

• It gets activated at the site of action by the breakdown of one or more peptide bonds

• Ex: chymotrypsin, pepsinogen and plasminogen

• Certain enzymes keeps interconverting from active to inactive and vice-versa depending on the need of body

• Ex: Glycogen phosphorylase, Phosphorylase b


Compartmentation

• The enzyme remains confined to particular area of cell/body which makes it exclusive

• For instance: fatty acid synthesis takes place in cytosol whereas fatty acid oxidation takes in mitochondriaOrganelle Enzyme/metabolic pathway

Cytoplasm Aminotransferase, peptidases, glycolysis, HMP shunt

Mitochondria Fatty acid oxidation, Kreb’s Cycle, Urea Cycle, ETC

Nucleus Biosynthesis of DNA and RNA

Endoplasmic Reticulum Protein Biosynthesis, Triacylglycrol and phospholipid synthesis

Lysosomes Lysozyme, phosphatases, phospholipases, hydrolases, proteases

Golgi Appartus Glucos-6 phosphatease, glucosyl and galactosyl transferase

Peroxisomes Catalases, Urea oxidase, D-amino acid oxidasewww.facebook.com/notesdental

Control of enzyme synthesis

• Most of the enzyme particularly the rate limiting ones are present in very low concentration

• Based on the amount of enzyme present in the body, enzymes are– Constitutive enzymes: its levels are not controlled

and it remain almost constant

– Adaptive enzymes: their level increases or decreases as per body needs

• Synthesis of enzyme are regulated by gene.

• Regulation by induction / repression


Enzyme Degradation

• Enzymes have their self-destructing capabilities.

• But it is highly variable and in general

– The key and regulatory enzyme are most rapidly degraded

– Not so important enzyme have longer half life

• Ex: LDH4 – 5-6 days, LDH1 – 8-12 hrs, amylase – 3-5 hrs


Isoenzyme

• When same reaction is catalyzed by two or more different molecular forms of an enzyme, it is called isoenzyme

• It may occur in the same species, in the same tissue, or even in the same cell.

• The different forms of the enzyme generally differ in kinetic or regulatory properties

• Ex: hexokinase - 4, lactate dehydrogenase (LDH) –5, creatinine phosphate (CPK) - 3 , creatininekinase (CK) - 3, Alkaline phosphate (ALP) – 6, Alcohol dehydrogenase (ADH) - 2


Diagnostic Importance of Enzyme

• Estimation of enzyme activities in biological fluid is of great clinical importance.

• The enzyme can be divided in 2 groups

– Plasma Specific or plasma functional enzyme

– Non-plasma specific or plasma non-functional enzyme


Plasma Specific• Present in the plasma normally and have specific

fucntion

• Their value is higher in plasma than tissue

• They are mainly synthesized in liver and enter the circulation

• Ex: Lipoprotein lipase, plasmin, thrombin, cholineesterase, ceruloplasmin

• Impairment of liver function or genetic disorder –leads to enzyme deficiency

• Wilson disease – deficiency of ceruloplasmin


Non-plasma specific

• These enzymes are present in the low level in plasma compared to the tissue

• Estimation of activities of these enzymes serves for the diagnosis and prognosis of several disease - markers of disease

• The raised enzyme level may indicate

– Cellular damage

– Increased rate of cell turnover

– Proliferation of cells

– Increased synthesis of enzymeswww.facebook.com/notesdental

Important Diagnostic Enzymes

• Amylase – Acute pancreatitis• Serum glutamate pyruvate transferase (SGPT) – liver

disease (hepatitis)• Serum glutamate oxaloacetate transaminase (SGOT) –

Heart attacks (myocardial infarction)• Alkaline phosphatase – Rickets, obstructive jaundice• Acid phophatase – cancer of prostate gland• Lactate dehydrogenase (LDH) – heart attacks, liver disease• Creatinine phosphokinase (CPK) – myocardial infarction• Aldolase – Muscular dystrophy


Amylase

• Activity increased in acute pancreatitis

• Normal level – 0.2-1.5 IU/l

• Peak value in 8-12 hrs – onset of disease and returns to normal in 3-4 days

• Urine analysis

• Serum analysis – chronic pancreatitis, acute parotitis (mumps) and obstruction of pancreatic duct


Serum glutamate pyruvatetransferase (SGPT)

• Also known as Alanine transaminase (ALT)

• Normal level – 3-4.0 IU/l

• Acute hepatitis of viral or toxic origin

• Jaundice and cirrohosis of liver


Serum glutamate oxaloacetatetransaminase (SGOT)

• Also known as Aspartate transaminase

• Normal 4-4.5 IU/l

• Increase in myocardial infarction and also in liver diseases

• SGPT is more specific for liver disease and SGOT for MI – SGPT more cytosomal enzyme while SGOT is cytosol and mitochondria


Alkaline phosphatase

• Elevated in bone and liver disease

• Normal : 25-90 IU/l

• Diagnosis for

– Rickets,

– Hyperparathyroidism,

– Carcinoma of bone

– Obstructive jaundice

– Paget’s Disease


Acid phophatase

• Normal : 0.5 -4 KA units/dl

• Increased in cancer of prostate gland and Paget’s Disease

• Good tumor marker


Lactate dehydrogenase (LDH)• At least five different isozymes

• Assess the timing and extent of heart damage due to myocardial infarction MI (heart attack)– 12 hrs of MI: blood level of total LDH increases, and

there is more LDH2 than LDH1

– 24 hrs of MI: more LDH1 than LDH2

Type Composition Location

LDH1 HHHH Heart and erythrocyte

LDH2 HHHM Heart and erythrocyte

LDH3 HHMM Brain and kidney

LDH4 HMMM Skeletal muscle and liver

LDH5 MMMM Skeletal muscle and liver


Creatinine phosphokinase (CPK)

• Normal : 10-50 IU/l

• Diagnosis of

– MI - Very early detection

– Muscular dystrophy

– Hypothyroidism

– Alcoholism


Aldolase

• Normal ; 2+6 IU/l

• Diagnosis of

– Muscular dystrophy

– Liver disease

– Myocardial infarction

– Myasthenia gravis

– Leukemia


References

• Biochemistry – U. Satyanarayan, U. Chakerpeni

• Color_Atlas_of_Biochemistry_2005

• Harpers_Biochemistry_26th_ed

• Lehninger Principles of Biochemistry, Fourth Edition - David L. Nelson, Michael M. Cox.


enzyme

Health & Medicine

enzyme enzymes

introduction enzymes

individual enzymes

o extracellular o enzymes

enzyme inhibitors

enzyme nomenclature

given enzyme

digestive enzyme