Download - Enzymology

DEAR STUDENTS DEAR STUDENTS ANSWERANSWER

““WE ARE THE CATALYSTS OF THE WE ARE THE CATALYSTS OF THE LIVING WORLD, INCREASE THE RATE LIVING WORLD, INCREASE THE RATE OF REACTION THOUSAND OF REACTION THOUSAND TIMES.WORK IN AQUOUS MEDIUM. TIMES.WORK IN AQUOUS MEDIUM. PROTEIN IN NATURE. AND IN ACTION PROTEIN IN NATURE. AND IN ACTION SPECIFIC, ACCURATE; BIG IN SIZE SPECIFIC, ACCURATE; BIG IN SIZE BUT WITH SMALL ACTIVE SITE; BUT WITH SMALL ACTIVE SITE; HIGHLY EXPLOITED FOR DISEASE HIGHLY EXPLOITED FOR DISEASE DIAGNOSIS IN LAB CENTRES AND DIAGNOSIS IN LAB CENTRES AND ALSO USED FOR TREATMENT.’’ALSO USED FOR TREATMENT.’’

ENZYMESENZYMES

DR.K.S.SODHI,M.DR.K.S.SODHI,M.DD..

PROFESSOR PROFESSOR

BIO-CHEMISTRYBIO-CHEMISTRYMMIMS&R MULLANA MMIMS&R MULLANA

AMBALA.AMBALA.

© 2007 Paul Billiet ODWS

The Chemicals of Living Cells

©The Wellcome Trust

HISTORY of EnzymesHISTORY of Enzymes

As early as the late 1700s and As early as the late 1700s and early 1800s, the digestion of early 1800s, the digestion of meat by stomach secretionsmeat by stomach secretions and and the conversion of starch to the conversion of starch to sugars by plant extracts and sugars by plant extracts and saliva were known. However, the saliva were known. However, the mechanism by which this mechanism by which this occurred had not been identified.occurred had not been identified.

ENZYMOLOGYENZYMOLOGYContribution of Scientists.Contribution of Scientists.Definitions.Definitions.Mode of Action of Enzymes.Mode of Action of Enzymes.Factors Influencing Enzyme Factors Influencing Enzyme

Activity.Activity.Enzyme Inhibition.Enzyme Inhibition.Regulation of Enzymes.Regulation of Enzymes.Diagnostic Importance of Enzymes.Diagnostic Importance of Enzymes.Therapeutic Use of Enzymes.Therapeutic Use of Enzymes.

DEFINITIONSDEFINITIONSHOLOENZYMES HOLOENZYMES ( APOENZYMES+CO ( APOENZYMES+CO ENZ.)ENZ.)APOENZYMES;APOENZYMES; SINGLE SINGLE POLYPEPTIDECHAIN,MORE THAN ONE POLYPEPTIDECHAIN,MORE THAN ONE CHAIN,MULTI-ENZYME COMPLEX.CHAIN,MULTI-ENZYME COMPLEX.Co-ENZYMESCo-ENZYMES: Non Protein (VITAMINS): Non Protein (VITAMINS)METAL-ACTIVATEDMETAL-ACTIVATED ENZYMES. ENZYMES.(Zn,Cu,Fe,Mg,K,Ca etc.)(Zn,Cu,Fe,Mg,K,Ca etc.)ZYMASE: ZYMASE: Active without Active without modificationmodificationZYMOGENSZYMOGENS: Pro Enzymes : Pro Enzymes eg.Trypsinogen to trypsineg.Trypsinogen to trypsin

ISO-ENZYMESISO-ENZYMES: Physically distinct : Physically distinct perform same function.perform same function.

RIBOZYMES: RIBOZYMES: Small ribonuclearSmall ribonuclear particles.particles.

ENDOENZYMESENDOENZYMES: Produced in the : Produced in the cell. Function inside the cell.cell. Function inside the cell.

EXOENZYMESEXOENZYMES: Produced inside : Produced inside the cell. Act outside the cell.the cell. Act outside the cell.

METALLO ENZYMESMETALLO ENZYMES: Contain metal : Contain metal ions as essential component.ions as essential component.

HOUSE KEEPING ENZYMESHOUSE KEEPING ENZYMES: Levels of : Levels of Enzymes can not be controlled. Always Enzymes can not be controlled. Always present in cell.present in cell.

ADAPTIVE ENZYMESADAPTIVE ENZYMES: Regulated by : Regulated by genes. Conc.increase or Decrease.genes. Conc.increase or Decrease.

KEY ENZYMESKEY ENZYMES :Regulatory eg HMG- :Regulatory eg HMG-CO.ACO.A

HYBRID ENZYMESHYBRID ENZYMES :Produced by :Produced by genetic fusion.genetic fusion.

COFACTORSCOFACTORS An additional non-An additional non-protein molecule that protein molecule that is needed by some is needed by some enzymes to help the enzymes to help the reactionreaction

Tightly bound Tightly bound cofactors are called cofactors are called prosthetic groupsprosthetic groups

Cofactors that are Cofactors that are bound and released bound and released easily are called easily are called coenzymescoenzymes

Many vitamins are Many vitamins are coenzymescoenzymes

Nitrogenase enzyme with Fe, Mo and ADP cofactors

A GOOD TEACHER A GOOD TEACHER IS ALWAYS A GOOD IS ALWAYS A GOOD

CATALYST IN CATALYST IN STUDENTS LIFE.STUDENTS LIFE.

ALWAYS A GOOD CATALYST IN STUDENTS LIFEALWAYS A GOOD CATALYST IN STUDENTS LIFE

DISTRIBUTION OF 17 HORSESDISTRIBUTION OF 17 HORSES

OLDMAN AND THREE SONS.OLDMAN AND THREE SONS.DISTRIBUTION OF HORSES.DISTRIBUTION OF HORSES.ELDER ½ELDER ½MIDDLE 1/3MIDDLE 1/3LITTLE 1/9LITTLE 1/9

Notice that without the enzyme it takes a lot more Notice that without the enzyme it takes a lot more energy for the reaction to occur. By lowering the energy for the reaction to occur. By lowering the

activation energy you speed up the reaction.activation energy you speed up the reaction.

Energy In ReactionsEnergy In Reactions Energy is released Energy is released

or absorbed or absorbed whenever chemical whenever chemical bonds are formed bonds are formed or broken.or broken.

Because chemical Because chemical reactions involve reactions involve breaking and breaking and forming of bonds, forming of bonds, they involve they involve changes in energy.changes in energy.

Enzymes as Biological CatalystsEnzymes as Biological Catalysts Enzymes are Enzymes are

proteins that proteins that increase the rate increase the rate of reaction by of reaction by lowering the lowering the energy of energy of activationactivation

They catalyze They catalyze nearly all the nearly all the chemical chemical reactions taking reactions taking place in the cells place in the cells of the bodyof the body

Enzymes have Enzymes have unique three-unique three-dimensional dimensional shapes that fit the shapes that fit the shapes of shapes of reactants reactants (substrates)(substrates)

Thermodynamics

The energies of various stages of a chemical reaction. Substrates need a large amount of energy to reach a transition state, which then decays into products.

The enzyme stabilizes the transition state, reducing the energy needed to form products.

As all catalysts, enzymes do not alter the position of the chemical equilibrium of the reaction. Usually, in the presence of an enzyme, the reaction runs in the same direction as it would without the enzyme, just more quickly.

For example, carbonic anhydrase catalyzes its reaction in either direction depending on the concentration of its reactants.

(in tissues; high CO2 concentration)

in lungs; low CO2 concentration).

Kinetics

Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products.

The enzyme (E) binds a substrate (S) and produces a product (P).

In 1902 Victor Henri contribute was to think of enzyme reactions in two stages. In the first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex.

This is sometimes called the Michaelis complex.

The enzyme then catalyzes the chemical step in the reaction and releases the product.

Saturation curve for an enzyme reaction showing the relation between the substrate concentration (S) and rate (v).

Enzyme rates depend on solution conditions and substrate concentration.

Conditions that denature the protein abolish enzyme activity, such as high temperatures, extremes of pH or high salt concentrations.

while raising substrate concentration tends to increase activity. Saturation happens because, as substrate concentration increases, more and more of the free enzyme is converted into the substrate-bound ES form.

At the maximum velocity (Vmax) of the enzyme, all the enzyme active sites are bound to substrate, and the amount of ES complex is the same as the total amount of enzyme. However, Vmax is only one kinetic constant of enzymes.

Km, : is the substrate concentration required for an enzyme to reach one-half its maximum velocity. Each enzyme has a characteristic Km for a given substrate.

kcat : is the number of substrate molecules handled by one active site per second.

So The efficiency of an enzyme = kcat/Km.

This is also called the specificity constant and incorporates the rate constants for all steps in the reaction (affinity and catalytic ability).

CO-ENZYMESCO-ENZYMESEssential for Biological activity.Essential for Biological activity.Low molecular weight, Organic in natureLow molecular weight, Organic in natureNon protein in nature.Non protein in nature. .Combine loosely with Enzyme &separate .Combine loosely with Enzyme &separate

later.later.Thermos table.Thermos table.Help in group transfer.Help in group transfer.Bind to apoenzymes.Bind to apoenzymes.GTP, NADP, FMN, FAD, Biotin, Lipoic Acid, GTP, NADP, FMN, FAD, Biotin, Lipoic Acid,

Pyridoxal Phosphate,etc. (Vitamins)Pyridoxal Phosphate,etc. (Vitamins)Co-enzyme separate from apo-Enz after Co-enzyme separate from apo-Enz after

reaction.reaction.Can be separated by Dialysis.Can be separated by Dialysis.

Co-Enzymes can be divided into two Co-Enzymes can be divided into two groups.groups.

A.Oxidoreductases.NADH.NADPH,FADA.Oxidoreductases.NADH.NADPH,FAD..

B. Transfer Groups.B. Transfer Groups.Thiamine-Hydroxyl group.Thiamine-Hydroxyl group.Pyridoxal phosphate-Amino groupPyridoxal phosphate-Amino groupTetrahydrofolate-one CarbonTetrahydrofolate-one CarbonBiotin-Carbon dioxideBiotin-Carbon dioxide..

Control Points of Gene Regulation

Prokaryotics

DNA

ribosomemRNA

proteins

Post-translationalcontrol Eukaryotics

proteins

cap5’ 3’

tail

mature mRNA

DNA

5’3’process

mRNA

Juang RH (2004) BCbasics

Translation

Activity

Proteolysis

Transcription

RNA ProcessingRNA Transport

RNA Degradation

Enzyme structureEnzyme structure Enzymes are Enzymes are

proteinsproteins

They have a They have a globular shapeglobular shape

A complex 3-D A complex 3-D structurestructure

© 2007 Paul Billiet ODWSHuman pancreatic amylase

STRUCTURESTRUCTURE1.MONOMERIC: Single Peptide.1.MONOMERIC: Single Peptide.

2.OLIGOMERIC: Many peptide Chains.2.OLIGOMERIC: Many peptide Chains.

3.Multienzyme Complex:3.Multienzyme Complex:

Fatty Acid SynthaseFatty Acid Synthase

LDH Complex.LDH Complex.

Prostaglandin Synthase Complex.Prostaglandin Synthase Complex.

ENZYMES UNITSENZYMES UNITSKINGARMSTRONG.KINGARMSTRONG.SOMOGY.SOMOGY.REITMAN FRANKEL.REITMAN FRANKEL.SPECTROPHOTOMETRIC.SPECTROPHOTOMETRIC.KATAL.KATAL.INTERNATIONAL UNIT.INTERNATIONAL UNIT.

ENZYMEZS ESTIMATED ENZYMEZS ESTIMATED FROM:FROM:

WHOLE BLOOD, SERUM, PLASMA.WHOLE BLOOD, SERUM, PLASMA.RED BLOOD CELLS.RED BLOOD CELLS.C.S.F.C.S.F.URINE.URINE.SWEAT.SWEAT.SALIVA.SALIVA.SEMEN.SEMEN.AMNIOTIC FLUID.AMNIOTIC FLUID.Tears.Tears.

TISSUES BRAIN,HEART,LIVER,KIDNEY,MUSCLE

MUSCLE→ ← HEART

→LIVER ←STOMACH

BRAIN

← KIDNEY

←INTESTINE

PLASMA ENZYMESPLASMA ENZYMESFUNCTIONALFUNCTIONAL PLAMSMA ENZYMES. PLAMSMA ENZYMES.

eg. LIPOPROTEIN LIPASE, BLOOD eg. LIPOPROTEIN LIPASE, BLOOD CLOT DISSOLVING ENZYMES etc.CLOT DISSOLVING ENZYMES etc.

NON FUNCTIONALNON FUNCTIONAL PLASMA PLASMA ENZYMES. eg: SGOT, ENZYMES. eg: SGOT, SGPT,AMYLASE,CPK,LDH,LIPASE,ACIDSGPT,AMYLASE,CPK,LDH,LIPASE,ACID-PHOSPHATASE,ALKALINE PHOS., -PHOSPHATASE,ALKALINE PHOS., CERULOPLASMIN etc.CERULOPLASMIN etc.

NATURE OF ENZYMESNATURE OF ENZYMES

Soluble, Colloidal, Organic Catalysts Soluble, Colloidal, Organic Catalysts

Formed by Living Cells ,Specific in Formed by Living Cells ,Specific in action, Protein In Nature ,Inactive at action, Protein In Nature ,Inactive at Zero degree centigrade ,Destroyed Zero degree centigrade ,Destroyed by moist heat at 100 degree by moist heat at 100 degree centigrade (Heat Labile), Huge in centigrade (Heat Labile), Huge in size, small Active Site, Used for size, small Active Site, Used for Treatment.Treatment.

DIFFERENCEDIFFERENCEBIO-CATALYSTBIO-CATALYST:: Enzymes, protein in nature Enzymes, protein in nature

except ribozymes, More specific, except ribozymes, More specific, more efficient and slight change in more efficient and slight change in structure alter its action.structure alter its action.

CATALYST:CATALYST: Inorganic, less sp., less efficient Inorganic, less sp., less efficient

and no change in structure.and no change in structure.

DNA, RNA, protein overview

DNA

RNA

Mutations

Amino acids, protein structure

Compartments of cell

COMPARTMENTATIONCOMPARTMENTATIONMITOCHONDRIA: MITOCHONDRIA: Enzymes of: E.T.C, Enzymes of: E.T.C,

TCA Cycle, Beta Oxidation, Urea Cycle, TCA Cycle, Beta Oxidation, Urea Cycle, Pyruvate to Acetyle Co-A.Pyruvate to Acetyle Co-A.

CYTOSOL: CYTOSOL: Glycolysis, HMP Shunt, Fatty Glycolysis, HMP Shunt, Fatty Acid Synthesis, Glucogenesis and Acid Synthesis, Glucogenesis and Glycogenolysis.Glycogenolysis.

NUCLEUS: NUCLEUS: DNA Synthesis, RNA DNA Synthesis, RNA Synthesis and Histones etc.Synthesis and Histones etc.

LYSOSOMES : Next SlideLYSOSOMES : Next Slide

FUNCTIONS OF ENZYMESFUNCTIONS OF ENZYMES

1. 1. Catalyse thousands of reactions.Catalyse thousands of reactions.

2. Digestive Enzymes help in igestion.2. Digestive Enzymes help in igestion.

3. Lysosomal Enzymes destroy in cell.3. Lysosomal Enzymes destroy in cell.

4. Lysozymes are bacteriocidal, local immunity4. Lysozymes are bacteriocidal, local immunity

(TEARS)(TEARS)

4. Detergents4. Detergents5. Textile.5. Textile.6. Leather Industry.6. Leather Industry.

What is a Ribozyme?

1) Enzyme2) Ribonucleic AcidNOT PROTEIN

1989 Nobel PrizeIn Chemistry

Sid Altman Tom Cech

RIBOZYMESRIBOZYMESSmall ribonucleic particles.Small ribonucleic particles.Contain rRNA.Contain rRNA.Highly substrate specific.Highly substrate specific.Used in Intron splicing,pre RNA to Used in Intron splicing,pre RNA to

RNA Peptidyl Transferase.RNA Peptidyl Transferase.Many ribozymes have hair-pin or Many ribozymes have hair-pin or

hammer head shaped active centre hammer head shaped active centre &require Divalent Mg++&require Divalent Mg++

Catalyse reaction on phosphpdiester Catalyse reaction on phosphpdiester bonds of other RNAbonds of other RNA

Ribozymes Have followingRibozymes Have following Drawbacks.Drawbacks.

Not as efficient as protein catalysts( In Not as efficient as protein catalysts( In RNA there are 4 nucleotides, in amino RNA there are 4 nucleotides, in amino acid are 20 in number.acid are 20 in number.

Act once only in chemical event,protein Act once only in chemical event,protein enzymes are reused several times.enzymes are reused several times.

Rate of catalytic activity is slower.Rate of catalytic activity is slower.Synthatic Ribozymes are having better Synthatic Ribozymes are having better

catalytic activity(Cleave infectious Virus)catalytic activity(Cleave infectious Virus)Used in Gene therapy.Used in Gene therapy.

ABZYMESABZYMESArtificially synthasized catalytic Artificially synthasized catalytic

antibodies against Enz. Sub. Complex antibodies against Enz. Sub. Complex in transition state of reaction. CATMAB in transition state of reaction. CATMAB (Catalytic Monoclonal Antibody).(Catalytic Monoclonal Antibody).

Sometimes natural abzymes are found Sometimes natural abzymes are found in blood,eg.antivasoactive intestinal in blood,eg.antivasoactive intestinal peptide autoantibodies.peptide autoantibodies.

Useful in diseases viz.abzyme against Useful in diseases viz.abzyme against gp120 envelop protein of HIV may gp120 envelop protein of HIV may prevent virus entry in to the host cell.prevent virus entry in to the host cell.

ANTIENZYME

Extracts of Intestinal Parasites like Ascaris,contain substances called antizymes,which inhibit the action of digestive enzymes pepsin and Trypsin.This is probably the reason why the worms are not digested in the Intestine.

Ribozyme vs. tRNAPhe

folding

The Future of Ribozymes

In Vitro Molecular Evolution of RNA

High Throughput Screening

Ribozyme-Based Therapies

+

In Clinical Trial...

HIV Gene Therapy...Bone Marrow Sample

Treat Stem Cells with Retroviral Vector

Re-Implant Treated Cells

Encodes Gene for anti-HIV Ribozyme

ACTIVE SITE OF ACTIVE SITE OF RIBONUCLEASESRIBONUCLEASES

It lies in a hydrophobic cleft.It lies in a hydrophobic cleft.77thth Lysine 41 Lysine 41stst Lysine on one Lysine on one

side and 12side and 12thth Histidine and Histidine and 119Histidine on the opposite 119Histidine on the opposite side.(URIDYLIC ACID)side.(URIDYLIC ACID)

Peptidyl transferase (chain Peptidyl transferase (chain Elongation)Elongation)

Removal of Introns.Removal of Introns.

The The SubstrateSubstrate

The substrate of an enzyme are The substrate of an enzyme are the the reactantsreactants that are that are activated by the enzymeactivated by the enzyme

Enzymes are Enzymes are specificspecific to their to their substratessubstrates

The specificity is determined by The specificity is determined by the the active siteactive site


PRODUCTPRODUCT

Substrate in the presence of Enzyme Substrate in the presence of Enzyme is converted in to product.is converted in to product.

The reaction can be Reversible or Ir-The reaction can be Reversible or Ir-reversible.reversible.

The increase in product The increase in product concentration can cause inhibition concentration can cause inhibition and stop the reaction in the forwaed and stop the reaction in the forwaed direction.direction.

ABBREVIATIONSABBREVIATIONS

ENZYME ENZYME [E][E]SUBSTRATE SUBSTRATE [S][S]PRODUCT PRODUCT [P][P]Enz. Sub. Complex Enz. Sub. Complex [ES][ES] INHIBITOR INHIBITOR [I][I]Enz.+Inh. Complex [Enz.+Inh. Complex [EIEI]]Enz.+Sub.+Inh. [ESI]Enz.+Sub.+Inh. [ESI]

Enzymes are Biological Enzymes are Biological CatalystsCatalysts

EnzymesEnzymes are proteins that: are proteins that: Increase the rate of reaction Increase the rate of reaction by lowering the energy of by lowering the energy of activation.activation.

Catalyze nearly all the Catalyze nearly all the chemical reactions taking chemical reactions taking place in the cells of the body.place in the cells of the body.

Have unique three-Have unique three-dimensional shapes that fit the dimensional shapes that fit the shapes of reactants. shapes of reactants.

Enzyme Deficiency

A variety of metabolic diseases are now known to be caused by deficiencies or malfunctions of enzymes.

Albinism, for example, is often caused by the absence of tyrosinase, an enzyme essential for the production of cellular pigments.

The hereditary lack of phenylalanine hydroxylase results in the disease phenylketonuria (PKU) which, if untreated, leads to severe mental retardation in children.

The The active siteactive site: : Is a region within an enzyme that Is a region within an enzyme that

fits the shape of molecules calledfits the shape of molecules called substratessubstrates. .

Contains amino acid R groups that Contains amino acid R groups that align and bind the substrate. align and bind the substrate.

Releases products when the Releases products when the reaction is complete.reaction is complete.

ACTIVE SITEACTIVE SITE

ACTIVE SITE OF ENZYMEACTIVE SITE OF ENZYME

Chymotrypsin Chymotrypsin His(57)Asp(102)Ser(195)His(57)Asp(102)Ser(195)

Trypsin Trypsin Histidine,SerineHistidine,Serine

Phosphoglucomutase SerinePhosphoglucomutase SerineCarboxypeptidase Carboxypeptidase

Histidine,Arginine,tyrosineHistidine,Arginine,tyrosineAldolase LysineAldolase Lysine

Active Site Avoids the Influence of Water

Preventing the influence of water sustains the formation of stable ionic bonds

-+

Active Site Is a Deep Buried Pocket

Why energy required to reach transition stateis lower in the active site?

It is a magic pocket

(1) Stabilizes transition(2) Expels water(3) Reactive groups(4) Coenzyme helps

(2)

(3)(4)

(1)CoE

+

-


ACTIVE SITEACTIVE SITE Generally the active site is situated Generally the active site is situated

on the cleft of the Enzyme.on the cleft of the Enzyme. Binding of substrate to active site Binding of substrate to active site

dependends upon the presence of sp. dependends upon the presence of sp. Groups or atoms at active site.Groups or atoms at active site.

During binding these groups,realign During binding these groups,realign themselves so as to fit the substrate.themselves so as to fit the substrate.

The substrate bind to active site by The substrate bind to active site by non co-valent bonds.(Hddrophobic in non co-valent bonds.(Hddrophobic in nature)nature)

Amino acid that make or break bonds Amino acid that make or break bonds called catalytic group.called catalytic group.

ACTIVE SITEACTIVE SITE

EnzymesEnzymesEnzymes provide a Enzymes provide a

site where site where reactants can be reactants can be brought together brought together to react.to react.

Such a site Such a site reduces the reduces the energy needed for energy needed for a reaction to a reaction to occur.occur.

All enzymes have an active site, where substrates are attracted

to.Enzymes are used over and over

again.

The Enzyme Substrate complex

When enzymes function the active site interacts with the substrate.

The active site shape matches the substrates shape.

Once the substrate and active site meet a change in shape of the active site causes a stress that changes the substrate and produces an end product.

Which one will fit ?

Enzymes may recognize and catalyze:Enzymes may recognize and catalyze: A single substrate.A single substrate. A group of similar substrates.A group of similar substrates. A particular type of bond. A particular type of bond.

ENZYME SPECIFICITYENZYME SPECIFICITY

MECHANISM OF MECHANISM OF ACTIONACTION

INDUCE FIT MODEL.INDUCE FIT MODEL.(KOSHLAND’S)(KOSHLAND’S)

LOCK AND KEY MODEL. LOCK AND KEY MODEL. (FISHER’S TEMPLATE THEORY)(FISHER’S TEMPLATE THEORY)

The Induced Fit The Induced Fit HypothesisHypothesis

Some proteins can change their shape Some proteins can change their shape (conformation)(conformation)

When a substrate combines with an When a substrate combines with an enzyme, it induces a change in the enzyme, it induces a change in the enzyme’s conformationenzyme’s conformation

TThe active site is then moulded into a he active site is then moulded into a precise conformationprecise conformation

Making the chemical environment Making the chemical environment suitable for the reactionsuitable for the reaction

The bonds of the substrate are stretched The bonds of the substrate are stretched to make the reaction easier (lowers to make the reaction easier (lowers activation energy)activation energy)


Induced-fit ModelInduced-fit ModelIn the In the induced-fit modelinduced-fit model of enzyme of enzyme

action:action: The active site is flexible, not rigid.The active site is flexible, not rigid. The shapes of the enzyme, active site, The shapes of the enzyme, active site,

and substrate adjust to maximum the and substrate adjust to maximum the fit, which improves catalysis.fit, which improves catalysis.

There is a greater range of substrate There is a greater range of substrate specificity. specificity.

The Lock and Key The Lock and Key HypothesisHypothesis

Fit between the substrate and the active Fit between the substrate and the active site of the enzyme is exact site of the enzyme is exact

Like a key fits into a lock very preciselyLike a key fits into a lock very precisely The key is analogous to the enzyme and the The key is analogous to the enzyme and the

substrate analogous to the lock.substrate analogous to the lock. Temporary structure called the enzyme-Temporary structure called the enzyme-

substrate complex formed substrate complex formed Products have a different shape from the Products have a different shape from the

substrate substrate Once formed, they are released from the Once formed, they are released from the

active site active site Leaving it free to become attached to Leaving it free to become attached to

another substrateanother substrate


Lock-and-Key ModelLock-and-Key ModelIn the In the lock-and-key modellock-and-key model of enzyme of enzyme

action: action: The active site has a rigid shape.The active site has a rigid shape. Only substrates with the matching Only substrates with the matching

shape can fit.shape can fit. The substrate is a key that fits the The substrate is a key that fits the

lock of the active site.lock of the active site. Rigid structure could not explain Rigid structure could not explain

flexibility shown by enzymesflexibility shown by enzymes

HO

H

Acid-Base Catalysis

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Induced to transition state

CO=

NH

HCH

NH

+

C- OOH

OH

-

+

HO

H

CO=

NH

HCH

CO=

NH

HCH

CO=

NH

HCH

Slow Fast Fast Very Fast

Acid-baseCatalysis Acid

catalysis

Basecatalysis

Both

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HO

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Specific

Basic Mechanism of Catalysis

3 basic types 1) Bond Strain2) Acid-base transfer3) Orientation

Conformational changeChemical reactionSpace arrangement

Carboxypeptidase ACarboxypeptidase BCarboxypeptidase Y

Concert

Sequential ChymotrypsinTrypsinElastase

non-polarRK

non-specific

YFWRKGA

Ser-proteaseEndopeptidase

Metal proteaseExopeptidase


Concerted Mechanism of Catalysis

1

2

3 4

5

O -

H+ H

COO -

(270)Glu

(248)Tyr

O -

H

His(196)

His (69)

Glu(72)

+Arg (145)

Carboxypeptidase A

C-terminus

ACTIVESITE

ACTIVESITE

Check forC-terminal

Site forspecificity

Activesite

pocket

Substratepeptide

chain

RNCN C

COO -

O -

C

+Zn

Jua

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RH

(2

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BC

ba

sics

MICHAELIS

CONSTANT(Km)

Salient Features of KmSalient Features of Km Km is sub. Conc.at ½ the max. velocityKm is sub. Conc.at ½ the max. velocity It denotes that 50% of Enzyme mol.are bound It denotes that 50% of Enzyme mol.are bound

with sub.at particular sub. Conc.with sub.at particular sub. Conc. Km is independent of Enzyme conc.If Enz. Km is independent of Enzyme conc.If Enz.

Conc. Is doubled, the Vmax will be double but Conc. Is doubled, the Vmax will be double but km will remain same.km will remain same.

Km is signature of Enzyme.Km is signature of Enzyme. Affinity of Enz. Towards its substrate is Affinity of Enz. Towards its substrate is

inversely related to the dissociation inversely related to the dissociation constant(smaller the dissociation greater the constant(smaller the dissociation greater the affinity.affinity.

Km denotes affinity of enzme for Km denotes affinity of enzme for substrate.lesser the Km more the affinity.substrate.lesser the Km more the affinity.

MICHAELIS CONSTANT MICHAELIS CONSTANT (Km)(Km)

It is defined as the conc. Of the It is defined as the conc. Of the substrate at which the reaction substrate at which the reaction velocity is half of the maximum velocity is half of the maximum velocity.velocity.

Km is independent of enzyme conc.Km is independent of enzyme conc.If an enzyme has a small value of If an enzyme has a small value of

Km, it achieves maximal catalytic Km, it achieves maximal catalytic efficency at low substrate conc.efficency at low substrate conc.

SIGNIFICANCESIGNIFICANCEGlucokinase has high Km is low Glucokinase has high Km is low

affinity for glucoseaffinity for glucose

Hexokinase has low Km, High affinity for Hexokinase has low Km, High affinity for Glucose ie glucose willGlucose ie glucose will

be provided to the vital organs even at be provided to the vital organs even at low glucose levels.low glucose levels.

Lab. Significance: The sub. Conc. Kept at Lab. Significance: The sub. Conc. Kept at saturation point at least 10 times the saturation point at least 10 times the Km so that reaction proceeds to Km so that reaction proceeds to completion.completion.

Clinical Significance: The Km value for Clinical Significance: The Km value for the given enzyme may differ from the given enzyme may differ from person to person and explains various person to person and explains various responses to drugs/chemicals.responses to drugs/chemicals.

The name of an enzyme:The name of an enzyme: Usually ends in Usually ends in –ase. –ase. Identifies the reacting substance. Identifies the reacting substance.

For example, For example, sucrasesucrase catalyzes the reaction of catalyzes the reaction of sucrose.sucrose.

Describes the function of the Describes the function of the enzyme. For example, enzyme. For example, oxidasesoxidases catalyze oxidation.catalyze oxidation.

Could be a common name, Could be a common name, particularly for the digestion particularly for the digestion enzymes such as enzymes such as pepsinpepsin and and trypsintrypsin..

Names of EnzymesNames of Enzymes

The top-level classification is: EC 1 Oxidoreductases: catalyze

oxidation/ reduction reactions . EC 2 Transferases: transfer a functional

group (e.g. a methyl or phosphate group). EC 3 Hydrolases: catalyze the hydrolysis

of various bonds . EC 4 Lyases: cleave various bonds by

means other than hydrolysis and oxidation.

EC 5 Isomerases: catalyze isomerization changes within a single molecule.

EC 6 Ligases: join two molecules with covalent bonds.

CLASSIFICATIONCLASSIFICATION

I.U.C.B.I.U.C.B. 1.1.OXIDO-REDUCTASEOXIDO-REDUCTASE.transfer of hydrogen .transfer of hydrogen

or addition of oxygen.Eg.LDHor addition of oxygen.Eg.LDH 2.2.TRANSFERASE.TRANSFERASE.Eg.Aminotransferase.Eg.Aminotransferase. Hexokinase.Hexokinase. 3.3.HYDROLASEHYDROLASE.Cleave bond adding water.Cleave bond adding water Eg. Acetyl choline esterase.Eg. Acetyl choline esterase. 4.4.LYASELYASE.Cleave without adding water .Cleave without adding water

(Aldolase)(Aldolase) 5.ISOMERASE.5.ISOMERASE. 6.6.LIGASE.LIGASE.Acetyl co-A Acetyl co-A

carboxylase,Glu.Synthatase,PRPP carboxylase,Glu.Synthatase,PRPP Synthatase.Synthatase.

Classification of Enzymes: Classification of Enzymes: Oxidoreductases and Oxidoreductases and

TransferasesTransferases

Classification: Hydrolases Classification: Hydrolases and Lyasesand Lyases

FACTORS AFFECTING FACTORS AFFECTING ENZYMEENZYME

1.SUBSTRATE CONCENTRATION.1.SUBSTRATE CONCENTRATION.2.ENZYME CONCENTRATION.2.ENZYME CONCENTRATION.3.TEMPERATURE.3.TEMPERATURE.4.pH.4.pH.5.EFFECT OF PRODUCT CONC.5.EFFECT OF PRODUCT CONC.6.PRESENCE OF ACTIVATORS6.PRESENCE OF ACTIVATORS7.INHIBITORS.7.INHIBITORS.8.EFFECT OF TIME.8.EFFECT OF TIME.

FACTORSFACTORS………………..………………..9.EFFECT OF CLOSE CONTCT.9.EFFECT OF CLOSE CONTCT.10.OXIDATION OF ADD.GROUPS.10.OXIDATION OF ADD.GROUPS.11.EFFECT OF LIGHT.11.EFFECT OF LIGHT.12.EFFECTS OF RADIATIONS.12.EFFECTS OF RADIATIONS.13.PRESENCE OF REPRESSOR 13.PRESENCE OF REPRESSOR

DEPRESSORDEPRESSOR14. ANTIZYMES.14. ANTIZYMES.

Substrate concentration: Non-Substrate concentration: Non-enzymic reactionsenzymic reactions

The increase in velocity is proportional to The increase in velocity is proportional to the substrate concentrationthe substrate concentration

Reaction velocity

Substrate concentration

Substrate ConcentrationSubstrate Concentration

The rate of The rate of reaction increases reaction increases as substrate as substrate concentration concentration increases (at increases (at constant enzyme constant enzyme concentration).concentration).

Maximum activity Maximum activity occurs when the occurs when the enzyme is enzyme is saturated.saturated.

Substrate concentration: Non-enzymic Substrate concentration: Non-enzymic reactionsreactions

The increase in velocity is proportional to The increase in velocity is proportional to the substrate concentrationthe substrate concentration

Reaction velocity


Substrate concentration: Substrate concentration: Enzymic reactionsEnzymic reactionswhen[ s] conc. Is increased velocity when[ s] conc. Is increased velocity

increases in the initial phase (Vmax.),but increases in the initial phase (Vmax.),but flatten afterward.flatten afterward.

Faster reaction but it reaches a saturation point Faster reaction but it reaches a saturation point when all the enzyme molecules are occupied.when all the enzyme molecules are occupied.

If you alter the concentration of the enzyme If you alter the concentration of the enzyme then Vthen Vmaxmax will change too. will change too.

Reaction velocity


Vmax


Enzyme ConcentrationEnzyme Concentration

The rate of reaction The rate of reaction increases as enzyme increases as enzyme concentration concentration increases (at increases (at constant substrate constant substrate concentration). concentration).

At higher enzyme At higher enzyme concentrations, concentrations, more substrate more substrate binds with binds with enzyme. enzyme.

End point reaction.End point reaction.

EFFECT OF EFFECT OF CONC.PRODUCTCONC.PRODUCT

At Equilibrium as per law of mass At Equilibrium as per law of mass action,the reaction rate is slowed action,the reaction rate is slowed down,it can slow,stopped or down,it can slow,stopped or reversed.reversed.

AA—E1——E1—BB—E2—≠— —E2—≠— CC—E3——E3—DD..

Increase in conc. Of D will cause feed Increase in conc. Of D will cause feed back Inhibition.back Inhibition.

The effect of temperatureThe effect of temperature For most enzymes the optimum For most enzymes the optimum

temperature is about 30°Ctemperature is about 30°C Many are a lot lower, Many are a lot lower,

cold water fish will die at 30°C because cold water fish will die at 30°C because their enzymes denaturetheir enzymes denature

A few bacteria have enzymes that can A few bacteria have enzymes that can withstand very high temperatures up to withstand very high temperatures up to 100°C100°C

Most enzymes however are fully Most enzymes however are fully denatured at 70°Cdenatured at 70°C


Affects of temperature on an Affects of temperature on an enzymeenzyme

If temp to high or If temp to high or to low the enzyme to low the enzyme will not fit. No will not fit. No reaction will occur.reaction will occur.

EnzymesEnzymes:: Are most active at an Are most active at an

optimum optimum temperature (usually temperature (usually 37°C in humans).37°C in humans).

Show little activity at Show little activity at low temperatures.low temperatures.

Lose activity at high Lose activity at high temperatures as temperatures as denaturation occurs.denaturation occurs.

Temperature and Enzyme Temperature and Enzyme ActionAction

The effect of temperatureThe effect of temperature

Temperature / °C

Enzyme activity

0 10 20 30 40 50

Q10 Denaturation

The effect of temperatureThe effect of temperature Q10 (Q10 (the temperature coefficientthe temperature coefficient) = the ) = the

increase in reaction rate with a 10°C rise in increase in reaction rate with a 10°C rise in temperature.temperature.

For chemical reactions the Q10 = 2 to 3For chemical reactions the Q10 = 2 to 3(the rate of the reaction doubles or triples (the rate of the reaction doubles or triples with every 10°C rise in temperature)with every 10°C rise in temperature)

Enzyme-controlled reactions follow this rule Enzyme-controlled reactions follow this rule as they are chemical reactionsas they are chemical reactions

BUT at high temperatures proteins BUT at high temperatures proteins denaturedenature

The optimum temperature for an enzyme The optimum temperature for an enzyme controlled reaction will be a balance controlled reaction will be a balance between the Q10 and denaturation.between the Q10 and denaturation.

Optimum pH Values

Most enzymes of the body have an optimum pH of about 7.4.

In certain organs, enzymes operate at lower and higher optimum pH values.

The effect of pHThe effect of pH Extreme pH levels will produce Extreme pH levels will produce denaturationdenaturation The structure of the enzyme is changed The structure of the enzyme is changed The active site is distorted and the substrate The active site is distorted and the substrate

molecules will no longer fit in itmolecules will no longer fit in it At pH values slightly different from the At pH values slightly different from the

enzyme’s optimum value, small changes in enzyme’s optimum value, small changes in the charges of the enzyme and it’s substrate the charges of the enzyme and it’s substrate molecules will occur molecules will occur

This change in ionisation will affect the binding This change in ionisation will affect the binding of the substrate with the active site.of the substrate with the active site.


How pH affects an enzyme

If the pH is to high or low the enzyme will not work, because its shape will change.

The effect of pHThe effect of pH

Optimum pH values

Enzyme activity Trypsin

Pepsin

pH

1 3 5 7 9 11


EnzymesEnzymes:: Are most active at Are most active at

optimum pH.optimum pH. Contain R groups of Contain R groups of

amino acids with amino acids with proper charges at proper charges at optimum pH.optimum pH.

Lose activity in low Lose activity in low or high pH as or high pH as tertiary structure is tertiary structure is disrupted.disrupted.

pH and Enzyme ActionpH and Enzyme Action

Optimum pH ValuesOptimum pH Values Most enzymes of the body have an Most enzymes of the body have an

optimum pH of about 7.4.optimum pH of about 7.4. In certain organs, enzymes operate at In certain organs, enzymes operate at

lower and higher optimum pH values.lower and higher optimum pH values.

ENZYME ACTIVATION BY ENZYME ACTIVATION BY INORGANIC IONSINORGANIC IONS

In the presence some inorganic ions In the presence some inorganic ions some enzymes show higher activity some enzymes show higher activity eg.Chloride ion activate salivary eg.Chloride ion activate salivary amylase,Ca. activates lipases.amylase,Ca. activates lipases.

Proenzymes in to enzymes.Proenzymes in to enzymes.Coagulatio factors are seen in blood Coagulatio factors are seen in blood

as zymogen.as zymogen.Compliment cascade,these Compliment cascade,these

activities needed occasionly.activities needed occasionly.

Enzyme InhibitionEnzyme InhibitionCompetitive Inhibtion.Competitive Inhibtion.Non-Competitive Inhibition.Non-Competitive Inhibition.Un-competitive Inhibition.Un-competitive Inhibition.Suicide Inhibition.Suicide Inhibition.Allosteric InhibitionAllosteric InhibitionKey EnzymesKey EnzymesFeedback Inhibition.Feedback Inhibition.Inducors.Glucokinase is induced by Inducors.Glucokinase is induced by

Insulin.Insulin.Repression (Heme is reprossor of ALA Repression (Heme is reprossor of ALA

Synthase.Synthase.

Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Ca

rtoo

n G

uid

eEq

uatio

n an

d De

scrip

tion

[II] binds to free [E] only,and competes with [S];increasing [S] overcomesInhibition by [II].

[II] binds to free [E] or [ES] complex; Increasing [S] cannot overcome [II] inhibition.

[II] binds to [ES] complex only, increasing [S] favorsthe inhibition by [II].

E + S → ES → E + P + II↓EII

←

↑

E + S → ES → E + P + + II II↓ ↓EII + S →EIIS

←

↑ ↑

E + S → ES → E + P + II ↓ EIIS

←

↑

EI

S X


Competitive Inhibition

Succinate Glutarate Malonate Oxalate

Succinate Dehydrogenase

Substrate Competitive InhibitorProduct

C-OO-

C-H C-H C-OO-

C-OO-

H-C-H H-C-H C-OO-

C-OO-

H-C-H H-C-H H-C-H C-OO-

C-OO-

C-OO-

C-OO-

H-C-H C-OO-

Sulfa Drug Is Competitive Inhibitor

-COOHH2N-

-SONH2H2N-

PrecursorFolicacid

Tetrahydro-folic acid

SulfanilamideSulfa drug (anti-inflammation)

Para-aminobenzoic acid (PABA)

Bacteria needs PABA for the biosynthesis of folic acid

Sulfa drugs has similar structure with PABA, andinhibit bacteria growth.

Domagk (1939)

Enzyme Inhibitors Are Extensively Used

●● Sulfa drug (anti-inflammation)

Pseudo substrate Pseudo substrate competitive inhibitor

●● Protease inhibitorPlaques in brain contains protein inhibitor

● HIV protease is critical to life cycle of HIV

HIV proteaseHIV protease (homodimer):(homodimer):

↑inhibitor is used to treat AIDS Symmetry

Notsymmetry

→ Human aspartyl protease:(monodimer)

domain 1

Asp Asp

domain 2

subunit 2

Asp

subunit 1

Asp

Alzheimer's disease

Signal Transduction Network (Ras vs. P53)

Cytosol

Cell membrane

Ras

Effectorenzyme

Signal protein

E2F Transcriptionfactor Target gene

mRNA

Inhibitor

P53

Cell division ON

Signal

Receptor

Nucleus

Ribosome

Transcription

Transcription

Apoptosis

Cell function are controlled by protein interactions

mRNA

Regulator protein


P

P

A

GP kinase

GP kinase

GP a

GP b

Glycogen synthase

Glycogen synthase P

Protein phosphatase-1

Protein phosphatase-1

Protein phosphatase inhibitor-1

Protein phosphatase inhibitor-1

Glycogen

PKA

P

active

inactive

PhosphataseG

luca

gon

Classification of Proteases

MetalProtease

SerineProtease

CysteineProtease

AspartylProtease

Carboxy-peptidase A

ChymotrypsinTrypsin

Papain

PepsinRenin

H57H57

D102D102

S195-OS195-O--

C25-SC25-S--

H195H195

D215D215

D32D32H2O

Non-specific

Non-specific

AromaticBasic

Non-polar

EDTAEGTA

DFPTLCKTPCK

PCMBLeupeptin

Pepstatin

Family Example Mechanism Specificity Inhibitor

E72E72 H69H69

Zn2+

H196H196


Sigm

oidal Curve E

ffect

Sigmoidal curve

Exaggeration of sigmoidal curveyields a drastic zigzag line that shows the On/Off point clearly

Positive effector (ATP)brings sigmoidal curveback to hyperbolic

Negative effector (CTP)keeps

Consequently, Allosteric enzyme can sense the concentration of the environment and adjust its activity

Noncooperative(Hyperbolic)

Cooperative(Sigmoidal)

CTPATP

vo

vo

[Substrate]Off On


INDUCTIONINDUCTIONInduction is effected through the Induction is effected through the

process of derepression.process of derepression.The inducer will relieve the repression The inducer will relieve the repression

on the operator site.on the operator site.In the absence of glucose,the enzymes In the absence of glucose,the enzymes

of Lactose metabolism will increase of Lactose metabolism will increase thousand times.thousand times.

Insulin is Inducer of Hexokinase Insulin is Inducer of Hexokinase Enzyme.Enzyme.

Barbiturates induce ALA Synthase.Barbiturates induce ALA Synthase.

REPRESSIONREPRESSION

Inhibition and repression Inhibition and repression reduce the Enzyme Velocity.reduce the Enzyme Velocity.

In case of Inhibition the In case of Inhibition the Inhibitor act directly on the Inhibitor act directly on the Enzyme.Enzyme.

Repressor acts at the gene Repressor acts at the gene level,effect is noticed after a level,effect is noticed after a lag period of Hours or Days.lag period of Hours or Days.

CO VALENT MODIFICATIONCO VALENT MODIFICATION

Activity of Enzyme can be Activity of Enzyme can be increased or decreasd by co-increased or decreasd by co-valent modifications Eg.Either valent modifications Eg.Either addition of group or Removal addition of group or Removal of groupof group

Zymogen activation by partial Zymogen activation by partial proteolysis is an Eg. Of co-proteolysis is an Eg. Of co-valent modificationvalent modification

ADP RIBOSYLATIONADP RIBOSYLATION

It is a type of co-valent modification.It is a type of co-valent modification. ADP-Ribose from NAD is added to ADP-Ribose from NAD is added to

enzyme/Protein.enzyme/Protein. ADP Ribosylation of Alfa Sub unit of G ADP Ribosylation of Alfa Sub unit of G

Protein leads to Inhibition of GTPase Protein leads to Inhibition of GTPase activity;hence G protein remains active.activity;hence G protein remains active.

Cholera toxin & Pertussive toxin act Cholera toxin & Pertussive toxin act through ADP-Ribosylation.through ADP-Ribosylation.

ADP Ribosylation of Glyeraldehyde 3P-ADP Ribosylation of Glyeraldehyde 3P-Dehydrosense,result in inhibition of Dehydrosense,result in inhibition of glycolysis.glycolysis.

xRegulatory

subunit

o

Regulation of Enzyme Activity

o xS I

x oS

Sx

S

oS

AA

Po R xR

+

III

or

inhibitor

proteolysis

phosphorylation

cAMP orcalmodulin

or

regulatoreffector

P

(-)

(+)

Inhibitor Proteolysis

Phosophorylation

Signal transduction

Feedback regulation

Jua

ng

RH

(2

00

4)

BC

ba

sics

REGULATION OF ENZYMESREGULATION OF ENZYMES

‘‘’’The action of enzymes can The action of enzymes can be activated or inhibited so be activated or inhibited so that the rate of enzyme that the rate of enzyme productin responds to the productin responds to the physiologcal need of the cell physiologcal need of the cell done to achieve cellular done to achieve cellular economy’’economy’’

1. Allosteric Regulation.1. Allosteric Regulation.2. Activation of Latent 2. Activation of Latent

Enzyme.Enzyme.3. Comprtmentation of 3. Comprtmentation of

Enzymes of different Enzymes of different Pathways.Pathways.

4. Control of Enzyme 4. Control of Enzyme Synthesis.Synthesis.

5. Enzyme Degradation.5. Enzyme Degradation.

CHANGE IN CATALYTICCHANGE IN CATALYTIC EFFICIENCY OF ENZYMEEFFICIENCY OF ENZYME

Catalytic effeciency is regulated is modulated Catalytic effeciency is regulated is modulated byby

A. Allosteric Regulation.A. Allosteric Regulation. B. Covalent modificationB. Covalent modification.. A. ALLOSTERIC REGULATION: Here the site is A. ALLOSTERIC REGULATION: Here the site is

different from substrate binding site, this site is different from substrate binding site, this site is called ALLOSTERIC SITE.called ALLOSTERIC SITE.

Low molecular wt. substances bind at site Low molecular wt. substances bind at site other than catalytic site,these are called other than catalytic site,these are called ALLOSTERIC MODULATORS.Location is called ALLOSTERIC MODULATORS.Location is called allosteric site.allosteric site.

Examples of SecondExamples of Second messengers are messengers are cAMP,cGMP and calcium etc.cAMP,cGMP and calcium etc.

These can change the enzyme These can change the enzyme conformation that may alter either conformation that may alter either Km or Vmax. Km or Vmax.

Based on this effect they are Based on this effect they are classified in to two classes.classified in to two classes.

1.K-class:Alter Km not Vmax.1.K-class:Alter Km not Vmax.2.V-class:Alter Vmax not Km.2.V-class:Alter Vmax not Km.

cAMP Controls Activity of Protein Kinase A

R C

R C

R

RA

A

A

A

AA

A

A

C

C

Regulatorysubunits

Catalyticsubunits

cAMPActive kinase

C

CREB

CREB

P

Nucleus

Activation

Geneexpression

ONDNA

Alb

ert

s e

t a

l (2

00

2)

Mo

lecu

lar

Bio

log

y o

f th

e C

ell

(4e

) p

. 8

57

, 8

58

EXAMPLE OF 2EXAMPLE OF 2ndnd MESSENGERMESSENGER

GLYCOGEN GLYCOGEN BREAKDOWBREAKDOWNN..

GLYCOGEN GLYCOGEN SYNTHESIS.SYNTHESIS.

A.Activator A.Activator A.InhibitorA.Inhibitor..

Allosteric Allosteric ActivatorActivator..

Hexokinase: ADPHexokinase: ADP Isocit.Dehydr. ADPIsocit.Dehydr. ADP Glu.Dehy. ADPGlu.Dehy. ADP Pyruvate Pyruvate

CarboxylaseCarboxylase Acetyl CoAAcetyl CoA

Allosteric Allosteric InhibitorInhibitor..

Glucose-6-P,ATPGlucose-6-P,ATP Glucose-6-P,ATPGlucose-6-P,ATP ATP, NADH.ATP, NADH.

ADPADP

HOMOTROPIC EFFECTHOMOTROPIC EFFECT: If the effector : If the effector Substace is substrate itself it is called Substace is substrate itself it is called

homotropic effect.homotropic effect.HETEROTROPIC EFFECTHETEROTROPIC EFFECT: Effector molecule : Effector molecule

is a substance other than substrate.is a substance other than substrate.SECOND MESSENGER:BindingSECOND MESSENGER:Binding of many of many

hormones to their surface receptor hormones to their surface receptor induce a change in enzyme catalysed induce a change in enzyme catalysed reaction by inducing the release of reaction by inducing the release of allosteric effector.These effector allosteric effector.These effector substances are called as 2substances are called as 2ndnd messenger messenger

Hormone is first messenger. Hormone is first messenger. Cont……Cont……

CONFERMATIONAL CHANGES CONFERMATIONAL CHANGES IN ALLOSTERIC ENZYMESIN ALLOSTERIC ENZYMES..

Most of Enzymes are oligomeric, Most of Enzymes are oligomeric, binding of effector moecule at the binding of effector moecule at the allosteric site brings a chage in the allosteric site brings a chage in the active site of enzyme leading to active site of enzyme leading to inhibition or activation.inhibition or activation.

Allosteric Enzyme exhist in two states.Allosteric Enzyme exhist in two states.A. Tense (T)A. Tense (T)B. Relaxed (R )B. Relaxed (R )

Both are in equlibrium.Both are in equlibrium.

CCC

Allosteric Enzyme ATCase

+

Active relaxed form

Inactive tense form

ATCase

RR

RR

RR

CCC

COO-

CH2

HN-C-COO-

H H-

---

OH2N-C-O-PO3

2-

= OH2N-C-

=

COO-

CH2

N-C-COO-

H H

---

-

Catalytic subunits

Catalytic subunits

Regulatory subunits

ATP

CTP

Nucleic acidmetabolism

Feedback inhibition

AspartateCarbamoylphosphate

Carbamoyl aspartate

CTP

CTP

CTP

CTP

CTP

CTP


Quaternary structure

The switch: Allosteric The switch: Allosteric inhibitioninhibition

Allosteric means Allosteric means “other “other site”site”

E

Active site

Allosteric site


The allosteric site the The allosteric site the enzyme “on-off” switchenzyme “on-off” switch

E

Active site

Allosteric site emptySubstrate

fits into the active site

The inhibitor molecule is

absent

Conformational change

Inhibitor fits into allosteric

site

Substratecannot fit into the active site

Inhibitor molecule is present

E


SH2domain

The Reception and Transduction of Signals

G protein

GDP

+ Signal

-GDP+GTP

GDP

GTP

GTP

Adenylate cyclase

+ Signal

ActivationP

ProteinPhosphatase

GlycogenSynthase

GlycogenSynthase P

active

Insulin

P P

PP kinase

Glucagon

A

G-protein-linked Receptor

Enzyme-linked ReceptozThe third group: Ion-channel-linked Receptor

Gilman, Rodbell (1994)

Glycogen breakdown

Glycogen

Jua

ng

RH

(2

00

7)

BC

ba

sics

Sigm

oidal Curve E

ffect

Sigmoidal curve

Exaggeration of sigmoidal curveyields a drastic zigzag line that shows the On/Off point clearly

Positive effector (ATP)brings sigmoidal curveback to hyperbolic

Negative effector (CTP)keeps

Consequently, Allosteric enzyme can sense the concentration of the environment and adjust its activity

Noncooperative(Hyperbolic)

Cooperative(Sigmoidal)

CTPATP

vo

vo

[Substrate]Off On


FEED BACK INHIBITIONFEED BACK INHIBITION

Enzyme is inhibited by end product Enzyme is inhibited by end product of reaction.of reaction.

A-B-C-D-E-F……….P.A-B-C-D-E-F……….P.P product will inhibit the enzyme P product will inhibit the enzyme

which converts A in to B.which converts A in to B.

COVALENT COVALENT MODIFICATIONSMODIFICATIONS

Two well known processesTwo well known processesA. PHOSPHORILATION.A. PHOSPHORILATION.B. PARTIAL PROTEOLYSIS.B. PARTIAL PROTEOLYSIS.A. Phosphorilation-A. Phosphorilation-

dephosphorilation:many enzymes are dephosphorilation:many enzymes are regulated by ATP dependent regulated by ATP dependent phosphorilation.Eg. Of phosphorilation.Eg. Of Serine,Threonine,and Serine,Threonine,and tyrosine,catalysed by protein kinases.tyrosine,catalysed by protein kinases.

PARTIAL PROTEOLYSISPARTIAL PROTEOLYSIS

Some enzymes are secreted as Some enzymes are secreted as inactive precursors called inactive precursors called Proenzymes or Zymogens.Proenzymes or Zymogens.

This convertion takes place as a This convertion takes place as a selective proteolysis.selective proteolysis.

It is ir-reversible processIt is ir-reversible processPepsinogen to pepsinPepsinogen to pepsinTrypsinogen to trypsin.Trypsinogen to trypsin.

Hexokinase have low KM High affinity Hexokinase have low KM High affinity for Glucose ie glucose will provide to for Glucose ie glucose will provide to the vital organs even at low glucose the vital organs even at low glucose levels.levels.

Lab. Significance: The sub. Conc. Kept Lab. Significance: The sub. Conc. Kept at saturation point at least 10 times the at saturation point at least 10 times the Km so that reaction proceeds to Km so that reaction proceeds to completion.completion.

Clinical Significance: The Km value for Clinical Significance: The Km value for the given enzyme may differ from the given enzyme may differ from person to person and explains various person to person and explains various response to drugs/chemicals.response to drugs/chemicals.

INHIBITORSINHIBITORS

InhibitorsInhibitors Inhibitors are chemicals that reduce the Inhibitors are chemicals that reduce the

rate of enzymic reactions. rate of enzymic reactions. The are usually specific and they work at The are usually specific and they work at

low concentrations.low concentrations. They block the enzyme but they do not They block the enzyme but they do not

usually destroy it. usually destroy it. Many drugs and poisons are inhibitors of Many drugs and poisons are inhibitors of

enzymes in the nervous system.enzymes in the nervous system.


The effect of enzyme The effect of enzyme inhibitioninhibition

Reversible inhibitors:Reversible inhibitors: These can These can be washed out of the solution of be washed out of the solution of enzyme by dialysis. enzyme by dialysis.

There are two categoriesThere are two categories

A.A. Competitive Inhibition.Competitive Inhibition.

B.B. Non Competitive Inhibition.Non Competitive Inhibition.


COMPETITIVE INHIBITION

There is close structural resemblance of Inhibitor with the Substrate.

Example:1.Malonate ions Inhibit Succinate

Dehydrgenae.2.Xanthene Oxidase is inhibited by

Allopurinol.


2.2. Non-competitive:Non-competitive: These are not influenced These are not influenced by the concentration of the substrate. It by the concentration of the substrate. It inhibits by binding irreversibly to the enzyme inhibits by binding irreversibly to the enzyme but but not at the active sitenot at the active site

Examples Examples Cyanide combines with the Iron in the Cyanide combines with the Iron in the

enzymes cytochrome oxidaseenzymes cytochrome oxidase Heavy metals, Heavy metals, AgAg or or HgHg, combine with , combine with –SH–SH

groups. groups.

These can be removed by using a chelating agent These can be removed by using a chelating agent such as EDTAsuch as EDTA



Irreversible inhibitors: Irreversible inhibitors: Combine Combine with the functional groups of the with the functional groups of the amino acids in the active site, amino acids in the active site, irreversiblyirreversibly

Examples:Examples: nerve gases and nerve gases and pesticides, containing pesticides, containing organophosphorus, combine with organophosphorus, combine with serine residues in the enzyme serine residues in the enzyme acetylcholine esteraseacetylcholine esterase


Enzyme Inhibition (Mechanism)

I

I

S

S

S I

I

I II

S

Competitive Non-competitive Uncompetitive

EE

Different siteCompete for

active siteInhibitor

Substrate

Ca

rtoo

n G

uid

eEq

uatio

n an

d De

scrip

tion

[II] binds to free [E] only,and competes with [S];increasing [S] overcomesInhibition by [II].

[II] binds to free [E] or [ES] complex; Increasing [S] cannot overcome [II] inhibition.

[II] binds to [ES] complex only, increasing [S] favorsthe inhibition by [II].

E + S → ES → E + P + II↓EII

←

↑

E + S → ES → E + P + + II II↓ ↓EII + S →EIIS

←

↑ ↑

E + S → ES → E + P + II ↓ EIIS

←

↑

EI

S X


Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Km’ [S], mM

vo

[S], mM

vo

II II

Km [S], mM

Vmax

II

Km’

Vmax’Vmax’

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax & Km decreased

II

1/[S]1/Km

1/vo

1/ Vmax

II

Two parallellines

II

Intersect at X axis

1/vo

1/ Vmax

1/[S]1/Km 1/[S]1/Km

1/ Vmax

1/vo

Intersect at Y axis

= Km’






C-OO-

C-H C-H C-OO-

C-OO-

H-C-H H-C-H C-OO-

C-OO-


C-OO-

C-OO-

C-OO-

H-C-H C-OO-

Applications of inhibitorsApplications of inhibitors

Negative feedbackNegative feedback: end point or : end point or end product inhibitionend product inhibition

PoisonsPoisons snake bite, plant alkaloids snake bite, plant alkaloids and nerve gasesand nerve gases

MedicineMedicine antibiotics, sulphonamides, antibiotics, sulphonamides, sedatives and stimulantssedatives and stimulants


Cell processes (e.g. respiration or photosynthesis) Cell processes (e.g. respiration or photosynthesis) consist of series of pathways controlled by enzymesconsist of series of pathways controlled by enzymes

AA BB CC DD EE FF

Enzyme pathwaysEnzyme pathways

eFeDeCeAeB

Each step is controlled by a different enzyme (eA, eB, eC etc)

This is possible because of enzyme specificity


End point inhibitionEnd point inhibition

The first step (controlled by The first step (controlled by eeAA) is often ) is often controlled by the end product (controlled by the end product (FF))

Therefore Therefore negative feedbacknegative feedback is possible is possible

AA BB CC DD EE FF

The end products are controlling their own The end products are controlling their own rate of productionrate of production

There is no build up of intermediates (B, C, There is no build up of intermediates (B, C, D and E)D and E)

eFeDeCeA eB

Inhibition


ATP is the end pointATP is the end point

This reaction lies near the beginning This reaction lies near the beginning of the respiration pathway in cellsof the respiration pathway in cells

The end product of respiration is The end product of respiration is ATPATP If there is a lot of ATP in the cell this If there is a lot of ATP in the cell this

enzyme is inhibitedenzyme is inhibited Respiration slows down and less ATP Respiration slows down and less ATP

is producedis produced As ATP is used up the inhibition stops As ATP is used up the inhibition stops

and the reaction speeds up againand the reaction speeds up again


The switch: Allosteric The switch: Allosteric inhibitioninhibition

Allosteric means Allosteric means “other “other site”site”

E

Active site

Allosteric site


Switching offSwitching off

These enzymes These enzymes have have two two receptor sitesreceptor sites

One site fits the One site fits the substrate like substrate like other enzymesother enzymes

The other site The other site fits an inhibitor fits an inhibitor moleculemolecule

Inhibitor fits into allosteric site


Inhibitor molecule


The allosteric site the The allosteric site the enzyme “on-off” switchenzyme “on-off” switch

E

Active site

Allosteric site emptySubstrate

fits into the active site

The inhibitor molecule is

absent

Conformational change

Inhibitor fits into allosteric

site


Inhibitor molecule is present

E


A change in shapeA change in shape

When the inhibitor is present it fits into When the inhibitor is present it fits into its site and there is a its site and there is a conformational conformational change change in the enzyme moleculein the enzyme molecule

The enzyme’s molecular shape changesThe enzyme’s molecular shape changes The The active siteactive site of the substrate of the substrate

changeschanges The substrate cannot bind with the The substrate cannot bind with the

substratesubstrate


Negative feedback is Negative feedback is achievedachieved

The reaction slows downThe reaction slows down This is This is notnot competitive inhibition but competitive inhibition but

it is reversibleit is reversible When the inhibitor concentration When the inhibitor concentration

diminishes the enzyme’s diminishes the enzyme’s conformation changes back to its conformation changes back to its active formactive form


PhosphofructokinasePhosphofructokinase The respiration pathway accelerates The respiration pathway accelerates

and ATP (the final product) builds up and ATP (the final product) builds up in the cellin the cell

As the ATP increases, more and more As the ATP increases, more and more ATP fits into the ATP fits into the allosteric siteallosteric site of the of the phosphofructokinase molecules phosphofructokinase molecules

The enzyme’s conformation changes The enzyme’s conformation changes again and stops accepting substrate again and stops accepting substrate molecules in the molecules in the active siteactive site

Respiration slows downRespiration slows down






Adapted from Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.49

C-OO-

C-H C-H C-OO-

C-OO-

H-C-H H-C-H C-OO-

C-OO-


C-OO-

C-OO-

C-OO-

H-C-H C-OO-

Sulfa Drug Is Competitive Inhibitor

-COOHH2N-

-SONH2H2N-

PrecursorFolicacid

Tetrahydro-folic acid

SulfanilamideSulfa drug (anti-inflammation)

Para-aminobenzoic acid (PABA)

Bacteria needs PABA for the biosynthesis of folic acid

Sulfa drugs has similar structure with PABA, andinhibit bacteria growth.

Domagk (1939)

Km

Enzyme Inhibition (Plots)

I II Competitive Non-competitive Uncompetitive

Dir

ect

Plo

tsD

ou

ble

Rec

ipro

cal

Vmax Vmax

Km Km’ [S], mM

vo

[S], mM

vo

II II

Km [S], mM

Vmax

II

Km’

Vmax’Vmax’

Vmax unchangedKm increased

Vmax decreasedKm unchanged

Both Vmax & Km decreased

II

1/[S]1/Km

1/vo

1/ Vmax

II

Two parallellines

II

Intersect at X axis

1/vo

1/ Vmax

1/[S]1/Km 1/[S]1/Km

1/ Vmax

1/vo

Intersect at Y axis

= Km’



Irreversible inhibitorsIrreversible inhibitors: : Combine with the functional groups Combine with the functional groups of the amino acids in the active site, of the amino acids in the active site, irreversibly.irreversibly.

Examples:Examples: nerve gases and pesticides, nerve gases and pesticides, containing organophosphorus, containing organophosphorus, combine with serine residues in the combine with serine residues in the enzyme acetylcholine esterase.enzyme acetylcholine esterase.



Reversible inhibitorsReversible inhibitors:: These can be washed out of the These can be washed out of the solution of enzyme by dialysis. solution of enzyme by dialysis.

There are two categories.There are two categories.


The effect of The effect of enzyminhibitionenzyminhibition

1.1. CompetitiveCompetitive: : These compete These compete with the substrate with the substrate molecules for the molecules for the active site.active site.

The inhibitor’s action The inhibitor’s action is proportional to is proportional to its concentration.its concentration.

Resembles the Resembles the substrate’s substrate’s structure closely.structure closely.

Enzyme inhibitor complex

Reversible

reaction

E + I EI


CLINICAL APPLICATIONS OF CLINICAL APPLICATIONS OF COMPETITVE INHIBITORSCOMPETITVE INHIBITORS

DRUGDRUG ENZYMEENZYME TRUE TRUE SUB.SUB.

Clinical Clinical App.App.

ALLOPURIALLOPURINOLNOL

XANTHINE XANTHINE OXIDASEOXIDASE

HYPOXANHYPOXANTHENETHENE

GOUTGOUT

SULFONASULFONAMIDEMIDE

Dihydro Dihydro pteroate pteroate SynthaseSynthase

PABAPABA ANTIBIOTIANTIBIOTICC

ETHANOLETHANOL Al.Dehy.Al.Dehy. METHANOMETHANOLL

METHANOMETHANOL L POISONINPOISONINGG

NON-COMPETITIVENON-COMPETITIVE2.2. Non-competitive:Non-competitive: These are not These are not

influenced by the concentration of the influenced by the concentration of the substrate. It inhibits by binding irreversibly substrate. It inhibits by binding irreversibly to the enzyme but not at the active site.to the enzyme but not at the active site.

Examples Examples Cyanide combines with the Iron in the Cyanide combines with the Iron in the

enzymes cytochrome oxidase.enzymes cytochrome oxidase. Heavy metals, Ag or Hg, combine with –SH Heavy metals, Ag or Hg, combine with –SH

groups. groups.

These can be removed by using a chelating These can be removed by using a chelating agent such as EDTA.agent such as EDTA.


Applications of inhibitorsApplications of inhibitors

Negative feedbackNegative feedback: end point or : end point or end product inhibitionend product inhibition

PoisonsPoisons snake bite, plant alkaloids snake bite, plant alkaloids and nerve gases.and nerve gases.

MedicineMedicine antibiotics, sulphonamides, antibiotics, sulphonamides, sedatives and stimulantssedatives and stimulants


ISOENZYMESOR

ISOZYMES

ISOENZYMESISOENZYMES Isoenzymes or Isozymes are physically Isoenzymes or Isozymes are physically

distinct form of same enzyme having same distinct form of same enzyme having same specificity, but are present in different specificity, but are present in different tissues of same organism, in different cell tissues of same organism, in different cell compartment.compartment.

Useful for diagnosing diseases of different Useful for diagnosing diseases of different organs.organs.

Homomultimer:All the units are same.Homomultimer:All the units are same.Heteromultimer:Sub units are different.These Heteromultimer:Sub units are different.These

are produced by different genes.are produced by different genes.

IDENTIFICATION OF IDENTIFICATION OF ISOZYMESISOZYMES1.Agar gel or PAGE.They have 1.Agar gel or PAGE.They have

different mobility.different mobility.2.Heat stability.2.Heat stability.3.Inhibitors.Isozymes may be 3.Inhibitors.Isozymes may be

sensative to different sensative to different inhibitors.eg.tartrate labile.inhibitors.eg.tartrate labile.

4. Km value or substrate specificity. 4. Km value or substrate specificity. Eg.Glucokinase has high Km and Eg.Glucokinase has high Km and Hexokinase has low Km for Glucose.Hexokinase has low Km for Glucose.

5.Co-Factors.Eg Mitochondrial 5.Co-Factors.Eg Mitochondrial isocitrate dehydrogenase is NAD isocitrate dehydrogenase is NAD dependent,Cytoplasmic isocitrate dependent,Cytoplasmic isocitrate dehydrogenase is NADP dehydrogenase is NADP dependent.dependent.

6. Localisation: Lactate 6. Localisation: Lactate DehydrogenaseH4 heart,M4 DehydrogenaseH4 heart,M4 Muscles.Muscles.7.Specific antibodies identify 7.Specific antibodies identify sp.Isozyme.sp.Isozyme.

Isoenzyme name

Composition

Composition

Present in

Elevated in

LDH1 ( H4) HHHH

Myocardium, RBC

myocardial infarction

LDH2 (H3M1) HHHM Myocardium, RBC

LDH3 (H2M2) HHMM Kidney, Skeletal muscle

LDH4 (H1M3) HMMM Kidney, Skeletal muscle

LDH5 (M4) MMMM Skeletal muscle, Liver

Skeletal muscle and liver diseases

Isoenzymes Isoenzymes

IsoenzymesIsoenzymes catalyze the catalyze the same reaction in same reaction in different tissues different tissues in the body.in the body.

Lactate Lactate dehydrogenase, dehydrogenase, which converts which converts lactate to lactate to pyruvate, (LDH) pyruvate, (LDH) consists of five consists of five isoenzymes.isoenzymes.

Isoenzyme name

Composition

Present in

Elevated in

CK-1 BB BrainCNS diseases

CK-2 MBMyocardium/ Heart

Acute myocardial infarction

CK-3 MM

Skeletal muscle, Myocardium

DIAGNOSTIC DIAGNOSTIC SIGNIFICANCSIGNIFICANC

EE

DISORDERS DIAGNOSED BY DISORDERS DIAGNOSED BY ENZYMESENZYMES

1) Cardiac 1) Cardiac Disorders.Disorders.

2) Hepatic 2) Hepatic Disorders.Disorders.

3) Skeletal Muscle 3) Skeletal Muscle Disorders. Disorders.

4) Bone Disorders.4) Bone Disorders.

5) Pancreatic 5) Pancreatic Disorders. Disorders.

6) Salivary gland 6) Salivary gland diseae (Mumps) diseae (Mumps)

7) Malignancies 7) Malignancies

Plasma enzymes are of two typesPlasma enzymes are of two types::

1.1. A small group of enzymes secreted into A small group of enzymes secreted into the blood by certain cells e.g.the blood by certain cells e.g.the liver secretes zymogens (inactive the liver secretes zymogens (inactive form of enzymes) of blood coagulation.form of enzymes) of blood coagulation.

2.2. FUNCTIONALFUNCTIONAL: Lipoprotein : Lipoprotein lipase,Pseudocholine estrase,blood lipase,Pseudocholine estrase,blood coagulation.coagulation.

3.3. NON FUNCTIONAL ENZYMES:NON FUNCTIONAL ENZYMES:

2.2. A large group of enzymes are A large group of enzymes are released from cells during normal cell released from cells during normal cell turnover.turnover.These enzymes function These enzymes function intracellularly (inside cells) and have intracellularly (inside cells) and have no function in the blood.no function in the blood.In healthy individuals, the blood In healthy individuals, the blood levels of these enzymes are constant, levels of these enzymes are constant, as the rate of release from damaged as the rate of release from damaged cells into blood is equal to the rate of cells into blood is equal to the rate of removal of enzymes from blood.removal of enzymes from blood.

Elevated enzyme Elevated enzyme activity in blood activity in blood indicates tissue indicates tissue damage (due to damage (due to increased release of increased release of intracellular enzymes).intracellular enzymes).

A. A. Plasma Enzymes as Plasma Enzymes as diagnostic toolsdiagnostic tools

Diseases that cause tissue damage Diseases that cause tissue damage result in increased release of result in increased release of intracellular enzymes into the intracellular enzymes into the plasma.plasma.

Determination of the level of these Determination of the level of these enzymes is used for diagnosis of enzymes is used for diagnosis of heart, liver, skeletal muscle, etc.heart, liver, skeletal muscle, etc.

The level of these enzymes in plasma The level of these enzymes in plasma correlates with the extent of tissue correlates with the extent of tissue damage.damage.

CREATINE KINASE(CPK OR CK)

Found in Heart, Skeltol Muscles,Brain small amounts are also found in lungs, thyroid and Adrenal glands.

Not found in RBC so haemolysis no effect.

NORMAL SERUM LEVELS:10-50 IU/L at 30 degree Centigrade.

CREATINE KINSE……………………

RAISED LEVELS ARE FOUND IN : 1.Myocardial Infarction. 2. Crushing Muscular Injury. 3. Damage to cardiac muscle (Any region) 4. Brain Injury. 5. Hypothyroidism. 6. Hypokalemia Highest level in 3-6,peak 24-30 hours

normaml in 3days.

ISOENZYMES OF (CPK)1.BB (CPK1) Tissue. is of origin is

Brain,Maximum Electrophoretic mobility, presence in blood is 0%.

MB (CPK2) Found in heart muscles, Intermediate electrophoretic mobility, presence in blood is 0-3%.

MM (CPK 3) Found in skeltol muscles,Least electrophoretic mobility, in blood its conc. Is 97-100 %.

Myocardial muscle is the only Myocardial muscle is the only tissue that contains high level tissue that contains high level of CK2 (MB) isoenzyme.of CK2 (MB) isoenzyme.

Appearance of CK2(MB) in Appearance of CK2(MB) in plasma is specific for heart plasma is specific for heart infarction.infarction.

Following an acute myocardial Following an acute myocardial infarction,CK2appears in infarction,CK2appears in plasma 4-8 hours following plasma 4-8 hours following onset of chest pain (peak is onset of chest pain (peak is reached after 24 hoursreached after 24 hours).).

ASPARTATE AMINO TRANSFERASE

AST (SGOT)SERUM GLUTAMATE OXALOACETATE TRANSFERASE

NORMAL LEVELS: 0-41 IU/LRises in 12 hours ,Peak levels 24

hoursReturns to normal 3-5 days.

ALANINE TRANSAMINASE (ALT)

ALT Highest conc. In Liver and next is skeltol muscles.

Raised levels are found in liver diseases and muscle disorders.

Marked elevation are found in acute hepatitis and other liver diseases.

The presence of increased levels of The presence of increased levels of some enzymes in plasma is some enzymes in plasma is diagnostic to damage of a particular diagnostic to damage of a particular tissue; tissue; e.g. The enzyme alanine e.g. The enzyme alanine

aminotransferaseaminotransferase (ALT) is (ALT) is abundant in the liver and the abundant in the liver and the appearance of elevated levels of ALT appearance of elevated levels of ALT in plasma indicates damage to the in plasma indicates damage to the liver.liver.

ALKALINE PHOSPHATASE (ALP)

Works at optimum pH 9.Highest conc. Are found in Liver,

Bone,Intestine and Placenta.Diagnosis of Bone and Liver PathologyMetastatic or Primary Malignant may

increase the enzyme activity.It has many Iso-enzymes. Cont…..

ISOENZYMES OF (ALP) 1. Alfa-1 ALP : Biliary canaliculi raised

activity shows obstructive jaundice. 2. Alfa-2 ALP Its levels rises in Hepatitis. 3.Pre Beta ALP: Bone cells, Bone diseases

raised levels are found. 4. Gama ALP :Found in Intestinal cells.

Levels rise in Ulcerative colitis. 5. Distinct Type: levels rise in

Lymphomas,Decrease in Chronic myl.Leuk. 6. Regan Isoenzyme:Cancer of lung,liver,

Gut.

GAMA GLTAMYL TRANSPEPTIDASE

(GGT)It is a sensative indicator of liver

diseases, especially of alcoholism.There are no other serum enzyme

abnormalities.

ACID PHOSPHATEExhists at pH 5-6.Diagnosis of Carcinoma of Prostate.Also found in RBC.Used as cancer Marker.

LACTATE DEHYDROGENASE (LDH)

Enzyme of anaerobic glycolysis.Liver,Myocardium, RBC.It is a tetramer made up of four units.These units can be separated by

electrolysis.There are two sub units (H&M)

IsoenzymesIsoenzymes



LDH (ISOENZYMES) LDH 1 Tetramer of

four units.30% in S.

LDH 2. 35% in Serum.

LDH 3. 20% LDH 4 10% LDH 5 5%

Moves fastest at pH8.6,myocardium ,RBC

Myocardium RBC .

Brain Kidney Skeltol

muscles ,Liver ----- Do -----

AMYLASE / LIPASEDigestive enzymes,exocrine

pancreas.Levels rise in Acute Pancreatitis.Patient present with severe abd.

Pain.Lipase levels are raised in Intestinal

infarction,Pertonitis or Perforation.

CHOLINESTRASESecreted by hepatic cells.Always present in serum.Metabolism of drugs cocaine and

succinyl-choline.

TRYPSINRaised levels of Trypsin in

plasma occurs during acute stage of PANCREATITIS

Along with Amylase and Lipase.It is a more reliable index of

Pancreatic disease rather than Amylase/Lipase

Intracellular Distribution of Intracellular Distribution of Diagnostic EnzymesDiagnostic Enzymes

LiveLiverr

HeaHeartrt

PancrPancreaseas

SalivSalivary ary GlanGlandsds

BoBonene

MusMusclecle

BiliBiliary ary TracTractt

ProstProstateate

LDLD55

ALTALTASTAST

LDLD11

ASASTT

CKCK

LPSLPSAMSAMS

AMSAMS ALALPP

CKCK ALPALPGGGGTT

ACPACP

DISORDERS DIAGNOSED BY DISORDERS DIAGNOSED BY ENZYMESENZYMES

1) Cardiac 1) Cardiac Disorders.Disorders.

2) Hepatic 2) Hepatic Disorders.Disorders.

3) Skeletal Muscle 3) Skeletal Muscle Disorders. Disorders.

4) Bone Disorders.4) Bone Disorders.

5) Pancreatic 5) Pancreatic Disorders. Disorders.

6) Salivary gland 6) Salivary gland diseae (Mumps) diseae (Mumps)

7) Malignancies 7) Malignancies

NAME OF THE ENZYME

Conditions in which level of activity in serum is elevated

Aspartate Amino transferase (AST)Serum glutamate-oxaloacetate transaminase (SGOT)

Myocardial infarction, Liver disease especially with liver cell damage

Alanine Amino transferase (ALT)Serum glutamate-pyruvate transaminase (SGPT)

Liver disease especially with liver cell damage

Alkaline Phosphatase (ALP)

Liver disease- biliary obstructionOsteoblastic bone disease-rickets

Acid Phosphatase (ACP)

Prostatic carcinoma

glutamyl Transferase ( GT)

Liver disorder like liver cirrhosis

Creatine kinase (CK)

Myocardial infarction and skeletal muscle disease(muscular dystrophy

Lactate Dehydrogenase (LDH)

Myocardial infarction, other diseases like liver disease.some blood diseases

Amylase Acute pancreatitis

Isoenzymes Isoenzymes



Diagnostic Significance Diagnostic Significance Enzymes Enzymes

The levels of The levels of diagnostic enzymes diagnostic enzymes determine the determine the amount of damage in amount of damage in tissues.tissues.

B. Isoenzymes and Heart B. Isoenzymes and Heart DiseasesDiseases

Isoenzymes (or isozymes) are a group of Isoenzymes (or isozymes) are a group of enzymes that catalyze the same reaction.enzymes that catalyze the same reaction.

However, these enzymes do not have the However, these enzymes do not have the same physical properties (as they differ in same physical properties (as they differ in amino acid sequence).amino acid sequence).

Thus, they differ in electrophoretic Thus, they differ in electrophoretic mobility.mobility.

The plasma level of certain isozymes of the The plasma level of certain isozymes of the enzyme Creatine kinase (CK) level is enzyme Creatine kinase (CK) level is determined in the diagnosis of myocardial determined in the diagnosis of myocardial infarction.infarction.

CARDIAC MARKERSCARDIAC MARKERSCPK (MB)CPK (MB)LDH (1)LDH (1)CARDIAC TROPONIN (I)&(T)CARDIAC TROPONIN (I)&(T)BRAIN NATRIURETIC PEPTIDEBRAIN NATRIURETIC PEPTIDE(Marker of Ventricular function)(Marker of Ventricular function)ASTASTALTALT

Abnormal Liver enzymes Abnormal Liver enzymes and/or LFTs: and/or LFTs:

work-up and diagnosiswork-up and diagnosis

LIVER LIVER MARKERSMARKERS

Liver TestsLiver TestsAST, ALTAST, ALTAlkaline PhosphataseAlkaline PhosphataseGGTGGTBilirubinBilirubinAlbuminAlbuminProtime/INRProtime/INR

True “liver function tests”

AST, ALTAST, ALTAspartate aminotransferase, Aspartate aminotransferase,

alanine aminotransferasealanine aminotransferaseEnzymes that are in the hepatocyte Enzymes that are in the hepatocyte

and function during gluconeogenesisand function during gluconeogenesisLeak out of the hepatocytes in times Leak out of the hepatocytes in times

of injury and can be measured in the of injury and can be measured in the serumserum

Normally present in serum at Normally present in serum at levels ~30-40 U/Llevels ~30-40 U/L

Alkaline PhosphataseAlkaline Phosphatase Exists in liver in membrane of hepatocyte Exists in liver in membrane of hepatocyte

where it lines the canaliculuswhere it lines the canaliculus Liver > bone > intestineLiver > bone > intestine PlacentaPlacenta Normally changes with ageNormally changes with age

50

100

150

200

250

300

350

400

5 15 25 35 45 55 65 75 85

Other cholestatic Other cholestatic enzymesenzymes

GGT: gamma-glutamyltransferaseGGT: gamma-glutamyltransferaseFound in hepatocytes and biliary epithelial Found in hepatocytes and biliary epithelial

cellscells5’ nucleotidase5’ nucleotidase

Both these enzymes can be used to Both these enzymes can be used to confirm alk phos elevation is coming confirm alk phos elevation is coming from liverfrom liver

GGT is also sensitive to alcohol ingestionGGT is also sensitive to alcohol ingestion

BilirubinBilirubinBreakdown product of hemeBreakdown product of heme

70-80% of normal production is from 70-80% of normal production is from breakdown of hemoglobin in senescent RBCbreakdown of hemoglobin in senescent RBC

Conjugation of bilirubin occurs in ER of Conjugation of bilirubin occurs in ER of hepatocyte, and conjugated bilirubin is hepatocyte, and conjugated bilirubin is then transported into bile (rate limiting then transported into bile (rate limiting step)step)

Almost 100% of bilirubin in healthy Almost 100% of bilirubin in healthy people is indirectpeople is indirect

AlbuminAlbumin Important plasma protein synthesized Important plasma protein synthesized

by the liverby the liverHalf-life 20 daysHalf-life 20 daysLevels <3 mg/dL should raise the Levels <3 mg/dL should raise the

suspicion of chronic liver diseasesuspicion of chronic liver disease***not specific for liver disease***not specific for liver disease

Also reduced in heavy alcohol Also reduced in heavy alcohol consumption, chronic inflammation, consumption, chronic inflammation, protein malnutritionprotein malnutrition

PROSTATE MARPROSTATE MAR

PSA (prostate SP.ANTIGEN.PSA (prostate SP.ANTIGEN.ACP (Acid Phosphatase)ACP (Acid Phosphatase)

MUSCLE MARKERMUSCLE MARKERCK (MM)CK (MM)AST (Aspartate Amino Transferase)AST (Aspartate Amino Transferase)ALD (Aldolase)ALD (Aldolase)

BONE MARKERBONE MARKER

ALP (Alkaline Phosphatase)ALP (Alkaline Phosphatase)

1.Cardiac Markers:

e.g. Acute Myocardial Infarction (AMI).

1) The myocardium becomes ischemic and

undergoes necrosis.

2) Cellular contents are released into the

circulation. Blood levels of the following enzymes increase:

ASTAST LDLD11 CKCK

22. Hepatic Disorders. Hepatic Disorders

a)a) Hepatocellular DisordersHepatocellular Disorders::

(1) Viral hepatitis: Hepatitis B & (1) Viral hepatitis: Hepatitis B & Hepatitis C.Hepatitis C.

(2) Toxic hepatitis: caused by chemicals (2) Toxic hepatitis: caused by chemicals & & Toxins (e.g aflatoxin, Asp. flavus) Toxins (e.g aflatoxin, Asp. flavus)

Increased levels of the following Increased levels of the following enzymes :enzymes :

ALTALT ASTAST LDLD55

b) b) Biliary tract disordersBiliary tract disorders::

The plasma levels of the following The plasma levels of the following enzymes increase: enzymes increase:

ALPALP GGTGGT

3. 3. Skeletal Muscle Skeletal Muscle DisordersDisorders

Muscle dystrophy.Muscle dystrophy. Muscle trauma.Muscle trauma. Muscle hypoxia.Muscle hypoxia. Frequent I.M Injections.Frequent I.M Injections. The plasma levels of the following enzymes The plasma levels of the following enzymes

increase:increase:

CKCK ASTAST

4. 4. Bone DisordersBone Disorders::

1)1) Paget’s Bone Disease: caused by Paget’s Bone Disease: caused by increased increased osteoclastic activity.osteoclastic activity.

2) Rickets2) Rickets

3) Osteomalacia:3) Osteomalacia: The plasma levels of the following The plasma levels of the following enzymeenzyme increase: increase: ALPALP

5. 5. Acute PancreatitisAcute Pancreatitis

The plasma levels of the following The plasma levels of the following enzymes increase:enzymes increase:

LipaseLipase AMSAMS

6. Salivary Gland 6. Salivary Gland Inflammation:Inflammation:

In Mumps:In Mumps:

The levels of The levels of -Amylase -Amylase (AMS)(AMS) is significantly is significantly increasedincreased

7. 7. MalignanciesMalignanciesa)a) Plasma (Acid phosphatase) Plasma (Acid phosphatase)

ACP ACP levels increase in: levels increase in:

• Prostatic carcinoma.Prostatic carcinoma.• Bone metastatic carcinoma Bone metastatic carcinoma

b) Plasma levels of Alkaline b) Plasma levels of Alkaline phosphatase (ALP) increase phosphatase (ALP) increase in:in:

• Pancreatic carcinoma.Pancreatic carcinoma.• Bile duct carcinoma.Bile duct carcinoma.• Liver metastasis.Liver metastasis.

c) Plasma levels of Total c) Plasma levels of Total Lactate Lactate dehydrogenase (LDH) dehydrogenase (LDH) increase in:increase in:

• LeukemiaLeukemia• Lymphomas.Lymphomas.• Liver metastasis.Liver metastasis.

ENZYMES TUMOUR MARKERS

B. Isoenzymes and Heart B. Isoenzymes and Heart DiseasesDiseases

Isoenzymes (or isozymes) are a group of Isoenzymes (or isozymes) are a group of enzymes that catalyze the same reaction.enzymes that catalyze the same reaction.

However, these enzymes do not have the However, these enzymes do not have the same physical properties (as they differ in same physical properties (as they differ in amino acid sequence).amino acid sequence).

Thus, they differ in electrophoretic Thus, they differ in electrophoretic mobility.mobility.

The plasma level of certain isozymes of the The plasma level of certain isozymes of the enzyme Creatine kinase (CK) level is enzyme Creatine kinase (CK) level is determined in the diagnosis of myocardial determined in the diagnosis of myocardial infarction.infarction.

Many isoenzymes contain Many isoenzymes contain different subunits in various different subunits in various combinations.combinations.

CK occurs in 3 isoenzymes, each CK occurs in 3 isoenzymes, each is a dimer composed of 2 is a dimer composed of 2 subunits (B & M): CK1 = BB, CK2 subunits (B & M): CK1 = BB, CK2 = MB and = MB and CK3 = MM, each CK isozyme CK3 = MM, each CK isozyme shows a characteristic shows a characteristic electrophoretic mobility.electrophoretic mobility.

Myocardial muscle is the only Myocardial muscle is the only tissue that contains high level tissue that contains high level of CK2 (MB) isoenzyme.of CK2 (MB) isoenzyme.

Appearance of CK2(MB) in Appearance of CK2(MB) in plasma is specific for heart plasma is specific for heart infarction.infarction.

Following an acute myocardial Following an acute myocardial infarction,CK2appears in infarction,CK2appears in plasma 4-8 hours following plasma 4-8 hours following onset of chest pain (peak is onset of chest pain (peak is reached after 24 hoursreached after 24 hours).).

Alkaline PhosphataseAlkaline Phosphatase1.Alfa1-ALP Liver1.Alfa1-ALP Liver2.Alfa2-ALP Liver (Heat Labile)2.Alfa2-ALP Liver (Heat Labile)3.Pre Beta-ALP (BONES)3.Pre Beta-ALP (BONES)4.Gama ALP (Ulcerative 4.Gama ALP (Ulcerative

Colitis)Colitis)5.Regan ALP (Bronchogenic 5.Regan ALP (Bronchogenic

cancer)cancer)

Sialic Acid ResiduesSialic Acid Residues

ENZYMES IN OTHER ENZYMES IN OTHER BODY FLUIDSBODY FLUIDS

Adenosine deaminase in Adenosine deaminase in pleural fluid :Elevated in pleural fluid :Elevated in Tuberculosis not in Tuberculosis not in Malignant effusion.Malignant effusion.

LDH; In CSF,Pleural fluid & LDH; In CSF,Pleural fluid & Ascitic Fluid.Ascitic Fluid.

Elevated levels in Elevated levels in MalignacyMalignacy..

Enzymes as Therapeutic Enzymes as Therapeutic AgentsAgents

Dissolving Streptokinase,Urokinase.Dissolving Streptokinase,Urokinase.Asparaginase used in some leukemias.Asparaginase used in some leukemias.Deoxyribonuclease is adminstered via Deoxyribonuclease is adminstered via

respiratory route to clear viscid respiratory route to clear viscid secretions in pt. of cystic fibrosis.secretions in pt. of cystic fibrosis.

Serratiopeptidase is used to minimise Serratiopeptidase is used to minimise edema in acute inflamatory conditions.edema in acute inflamatory conditions.

Hyaluronidase for hypovolumiaHyaluronidase for hypovolumiaHemocoagulase used as hemostat.Hemocoagulase used as hemostat.

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ENZYMES USED IN ENZYMES USED IN DIAGNOSTICS PROCEDURESDIAGNOSTICS PROCEDURES

Urease Urea.Urease Urea.Uricase Uric Acid.Uricase Uric Acid.Glucose Oxidase Glucose.Glucose Oxidase Glucose.Peroxidase Cholesterol.Peroxidase Cholesterol.Hexokinase Glucose.Hexokinase Glucose.Lipase Triglycerides.Lipase Triglycerides.Alkaline phosphatase ELISA.Alkaline phosphatase ELISA.Restriction endonuclease RFLPRestriction endonuclease RFLP

Clinical Enzymology Questions

1. For a biological process to occur a free energy of activation must be overcome. Enzymes work in this process to:

a. Lower the free energy of activation b. Raise the free energy of activation c. Enzymes have no effect on free energy of activation d. The effect on free energy of activation is dependent on the

enzyme in question e. None of the above

2. CK-M and CK-B are examples of what type of enzyme?

a. Homogeneous enzymes b. Isoenzymes c. Heterogeneous enzymes d. Co-factors e. None of the above

3. A 68-year-old male presents to the emergency room with acute mental

confusion. Upon questioning his family members they recall that for the last several months he has been complaining of tingling and loss of feeling in his hands and feet, difficulty walking, and vomiting. Which of the following co-factors is he most likely suffering from a deficiency in?

a. Folic Acid coenzymes b. Biotin c. Flavin coenzymes d. Thiamine pyrophosphate e. B12 coenzymes

4. Which of the following type of enzyme reaction does not normally

require the use of a cofactor?

a. Oxidation-reduction reaction b. Group Transfer reactions c. Isomerizations d. Hydrolytic reactions

Fungal Diastase Fungal Diastase &Pepsin used as &Pepsin used as digestive enz.digestive enz.Ribozymes &AbzymesRibozymes &AbzymesStreptodornase; DNA Streptodornase; DNA applied locally.applied locally.Alpha-1-ant-trypsin; Alpha-1-ant-trypsin; EmphysemaEmphysema

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