(199)enzyme 2011

ENZYMOLOGY

Contribution of Scientists. Definitions. Mode of Action of Enzymes. Factors Influencing Enzyme Activity. Enzyme Inhibition. Regulation of Enzymes. Diagnostic Importance of Enzymes. Therapeutic Use of Enzymes.

ENZYMES

© 2007 Paul Billiet ODWS

BERZELIUS 1835 Starch. Hydrolysis.

KUHNE 1878

Enzyme mean yeast.

EDWARD BUCHNER

Sucrose to Ethanol

ENZYMES


EDWARD BUCHNER N.P.1907

ARTHUR HARDEN N.P. 1929

JAMES SUMNER N.P. 1946

JOHAN NORTHROP N.P. 1946

WILHELM OSTWALD N.P. 1909

WARBURG N.P. 1970

ENZYMES

DR.K.S.SODHI,M.D.

PROFESSOR

BIO-CHEMISTRY

MMIMS&R MULLANA AMBALA.


A GOOD TEACHER IS ALWAYS A GOOD

CATALYST IN STUDENTS LIFE.

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

DISTRIBUTION OF 17 HORSES OLDMAN AND THREE SONS. DISTRIBUTION OF HORSES. ELDER ½ MIDDLE 1/3 LITTLE 1/9

ENZYMES


EDWARD BUCHNER N.P.1907

ARTHUR HARDEN N.P. 1929

JAMES SUMNER N.P. 1946

JOHAN NORTHROP N.P. 1946

WILHELM OSTWALD N.P. 1909

WARBURG N.P. 1970

Vitro and Vivo Reactions

Starch into glucose. Boil for 2 hours in conc Hcl

For digesting meat need strong acid and long time

Starch is converted in to glucose in Minutes in the presence of enzymes.

With in hour at pH 7.4

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

ISO-ENZYMES: Physically distinct perform same function.

RIBOZYMES: Small ribonuclear particles. ENDOENZYMES: Produced in the cell.

Function inside the cell. EXOENZYMES: Produced inside the cell.

Act outside the cell.

METALLO ENZYMES: Contain metal ions as essential component.

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

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

KEY ENZYMES :Regulatory eg HMG-CO.A HYBRID ENZYMES :Produced by genetic fusion.

COFACTORS An additional non-protein

molecule that is needed by some enzymes to help the reaction

Tightly bound cofactors are called prosthetic groups

Cofactors that are bound and released easily are called coenzymes

Many vitamins are coenzymes

Nitrogenase enzyme with Fe, Mo and ADP cofactors

CO-ENZYMES Essential for Biological activity. Low molecular weight, Organic in nature Non protein in nature. .Combine loosly with Enzyme &separate later. Thermostable. Help in group transfer. Bind to apoenzymes. Eg.NAD, NADP, FMN, FAD, Biotin, Lipoic Acid,

Pyridoxal Phosphate,etc. (Vitamins) Co-enzyme separate from apo-Enz after reaction. Can be separated by Dialysis.

Co-Enzymes can be divided into two groups. A.Oxidoreductases.NADH.NADPH,FAD.

B. Transfer Groups. Thiamine-Hydroxyl group. Pyridoxal phosphate-Amino group Tetrahydrofolate-one Carbon Biotin-Carbon dioxide.

TABULAR FORM SHOWING CO.E

Enzyme structure Enzymes are proteins

They have a globular shape

A complex 3-D structure

© 2007 Paul Billiet ODWSHuman pancreatic amylase

STRUCTURE1.MONOMERIC: Single Peptide.

2.OLIGOMERIC: Many peptide Chains.

3.Multienzyme Complex:

Fatty Acid Synthase

LDH Complex.

Prostaglandin Synthase Complex.

ENZYMES UNITS KINGARMSTRONG. SOMOGY. REITMAN FRANKEL. SPECTROPHOTOMETRIC. KATAL. INTERNATIONAL UNIT.

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

PLASMA ENZYMES FUNCTIONAL PLAMSMA ENZYMES. eg.

LIPOPROTEIN LIPASE, BLOOD CLOT DISSOLVING ENZYMES etc.

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

NATURE OF ENZYMES

Soluble, Colloidal, Organic Catalysts Formed by Living Cells ,Specific in

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

DIFFERENCE BIO-CATALYST:

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

CATALYST:

Inorganic, less sp., less efficient and no change in structure.

THE ENZYMES SPEAK

“WE ARE THE CATALYSTS OF THE LIVING WORLD? PROTEIN IN NATURE, AND IN ACTION. SPECIFIC, RAPID AND ACCURATE; HUGE IN SIZE BUT WITH SMALL ACTIVE CENTRE; HIGHLY EXPLOITED FOR DISEASE DIAGNOSIS IN LAB CENTRES AND ALSO USED FOR TREATMENT.’’

TISSUES BRAIN,HEART,LIVER,KIDNEY,MUSCLE

MUSCLE→ ← HEART

→LIVER ←STOMACH

BRAIN

← KIDNEY

←INTESTINE

COMPARTMENTATION

MITOCHONDRIA: Enzymes of: E.T.C, TCA Cycle, Beta Oxidation, Urea Cycle,

Pyruvate to Acetyle Co-A. CYTOSOL: Glycolysis, HMP Shunt, Fatty

Acid Synthesis, Glucogenesis and Glycogenolysis.

NUCLEUS: DNA Synthesis, RNA Synthesis and Histones etc.

LYSOSOMES :

FUNCTIONS OF ENZYMES

1. Catalyse thousands of reactions. 2. Digestive Enzymes help in Digestion. 3. Lysosomal Enzymes destroy in cell. 4. Lysozymes are bacteriocidal, local

immunity. (TEARS) 4. Detergents 5. Textile. 6. Leather Industry.

What is a Ribozyme?

1) Enzyme

2) Ribonucleic Acid

NOT PROTEIN

1989 Nobel PrizeIn Chemistry

Sid Altman Tom Cech

RIBOZYMES

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

Transferase. Many ribozymes have hair-pin or hammer head

shaped active centre &require Divalent Mg++ Catalyse reaction on phosphpdiester bonds of other

RNA

Ribozymes Have following Drawbacks. Not as efficient as protein catalysts( In RNA

there are 4 nucleotides, in amino acid are 20 in number.

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

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

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

ABZYMES

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

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

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

Structure : As with proteins, we consider...

Primary: GGCCGAACUGGUA

Secondary:

Tertiary:

Secondary StructureWatson-Crick Base Pairing Helix Formation

B-DNA RNA

RNA usually assumesA-form helices…

Small pore along helical axis

“Rungs” stack obliquely to axis

A-DNA

Secondary Structure

Conserved base-pairing interactions result in...

• Three “stem” regions

• Uridine-containing turn

• An “augmenting helix” joining stems II and III

Ribozyme vs. tRNAPhe

folding

Tertiary Structure

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 RIBONUCLEASES It lies in a hydrophobic cleft. 7th Lysine 41st Lysine on one side and 12th

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

Peptidyl transferase (chain Elongation) Removal of Introns.

The Substrate

The substrate of an enzyme are the reactants that are activated by the enzyme

Enzymes are specific to their substrates The specificity is determined by the active

site


PRODUCT

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

The reaction can be Reversible or Ir-reversible.

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

ABBREVIATIONS

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

Enzyme Stabilizes Transition State

S

P

ES

EST

EP

ST

Reaction direction

Energy change

Energy required (no

catalysis)

Energy decreases (under

catalysis)

Sub.(S) Prod. (P)Enz(E)T = Transition stateV=rate of change of S to P/mt.

Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166

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

ACTIVE SITE OF ENZYME

Chymotrypsin His(57)Asp(102)Ser(195) Trypsin Histidine,Serine Phosphoglucomutase Serine Carboxypeptidase

Histidine,Arginine,tyrosine Aldolase 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

+

-


The active site: Is a region within an

enzyme that fits the shape of molecules called substrates.

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

Releases products when the reaction is complete.

Active Site

ACTIVE SITE

Generally the active site is situated on the cleft of the Enzyme.

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

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

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

Amino acid that make or break bonds called catalytic group.

ACTIVE SITE

MECHANISM OF ACTION

INDUCE FIT MODEL.(KOSHLAND’S) LOCK AND KEY MODEL. (FISHER’S

TEMPLATE THEORY)

The Induced Fit Hypothesis Some proteins can change their shape

(conformation) When a substrate combines with an enzyme, it

induces a change in the enzyme’s conformation The active site is then moulded into a precise

conformation Making the chemical environment suitable for the

reaction The bonds of the substrate are stretched to make

the reaction easier (lowers activation energy)


Induced-fit Model

In the induced-fit model of enzyme action: The active site is flexible, not rigid. The shapes of the enzyme, active site, and

substrate adjust to maximum the fit, which improves catalysis.

There is a greater range of substrate specificity.

The Lock and Key Hypothesis Fit between the substrate and the active site of the enzyme is

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

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

formed Products have a different shape from the substrate Once formed, they are released from the active site Leaving it free to become attached to another substrate


Lock-and-Key ModelIn the lock-and-key model of enzyme action: The active site has a rigid shape. Only substrates with the matching shape can fit. The substrate is a key that fits the lock of the

active site. Rigid structure could not explain flexibility

shown by enzymes

CLASSIFICATION 1.OXIDO-REDUCTASE.transfer of hydrogen or

addition of oxygen.Eg.LDH 2.TRANSFERASE.Eg.Aminotransferase. Hexokinase. 3.HYDROLASE.Cleave bond adding water Eg. Acetyl choline estrase. 4.LYASE.Cleave without adding water (Aldolase) 5.ISOMERASE. 6.LIGASE.Acetyl co-A

carboxylase,Glu.Synthatase,PRPP Synthatase.

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

FACTORS………………..

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

DEPRESSOR 14. ANTIZYMES.

Substrate concentration: Non-enzymic reactions

The increase in velocity is proportional to the substrate concentration

Reaction velocity

Substrate concentration

Substrate Concentration

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

Maximum activity occurs when the enzyme is saturated.

Substrate concentration: Enzymic reactionswhen[ s] conc. Is increased velocity increases in the initial phase (Vmax.),but flatten afterward.

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

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

Reaction velocity

Substrate concentration

Vmax


Salient Features of Km

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

particular sub. Conc. Km is independent of Enzyme conc.If Enz. Conc. Is doubled, the

Vmax will be double but km will remain same. Km is signature of Enzyme. Affinity of Enz. Towards its substrate is inversely related to the

dissociation constant(smaller the dissociation greater the affinity. Km denotes affinity of enzme for substrate.lesser the Km more

the affinity.

Enzyme Concentration

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

At higher enzyme concentrations, more substrate binds with enzyme.

End point reaction.

The effect of temperature

For most enzymes the optimum temperature is about 30°C

Many are a lot lower, cold water fish will die at 30°C because their enzymes denature

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

Most enzymes however are fully denatured at 70°C


Enzymes: Are most active at an

optimum temperature (usually 37°C in humans).

Show little activity at low temperatures.

Lose activity at high temperatures as denaturation occurs.

Temperature and Enzyme Action


Temperature / °C

Enzyme activity

0 10 20 30 40 50

Q10 Denaturation


Q10 (the temperature coefficient) = the increase in reaction rate with a 10°C rise in temperature.

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

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

BUT at high temperatures proteins denature The optimum temperature for an enzyme controlled

reaction will be a balance between the Q10 and denaturation.

The effect of pH

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

molecules will no longer fit in it At pH values slightly different from the enzyme’s

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

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


The effect of pH

Optimum pH values

Enzyme activity Trypsin

Pepsin

pH

1 3 5 7 9 11


Enzymes: Are most active at

optimum pH. Contain R groups of

amino acids with proper charges at optimum pH.

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

pH and Enzyme Action

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.

ENZYME ACTIVATION BY INORGANIC IONS In the presence some inorganic ions some

enzymes show higher activity eg.Chloride ion activate salivary amylase,Ca. activates lipases.

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

zymogen. Compliment cascade,these activities needed

occasionly.

Enzyme Inhibition

Competitive Inhibtion. Non-Competitive Inhibition. Un-competitive Inhibition. Suicide Inhibition. Allosteric Inhibition Key Enzymes Feedback Inhibition. Inducors.Glucokinase is induced by Insulin. Repression (Heme is reprossor of ALA Synthase.

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


EFFECT OF CONC.PRODUCT At Equilibrium as per law of mass action,the

reaction rate is slowed down,it can slow,stopped or reversed.

A—E1—B—E2—≠— C—E3—D.

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

INDUCTION

Induction is effected through the process of derepression.

The inducer will relieve the repression on the operator site.

In the absence of glucose,the enzymes of Lactose metabolism will increase thousand times.

Insulin is Inducer of Hexokinase Enzyme. Barbiturates induce ALA Synthase.

REPRESSION

Inhibition and repression reduce the Enzyme Velocity.

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

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

CO VALENT MODIFICATION Activity of Enzyme can be increased or

decreasd by co-valent modifications Eg.Either addition of group or Removal of group

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

ADP RIBOSYLATION

It is a type of co-valent modification. ADP-Ribose from NAD is added to enzyme/Protein. ADP Ribosylation of Alfa Sub unit of G Protein leads

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

Cholera toxin & Pertussive toxin act through ADP-Ribosylation.

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

STABILIZATION

Enzyme molecules undergo usual wear & tear finally get degraded.Enzymes having thio (SH) groups eg Papian,Succinate dehydrogenase are stablized by glutathione. (G-SH).

Phosphofructokinase is stablized by growth hormone.

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

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REGULATION OF ENZYMES ‘’The action of enzymes can be

activated or inhibited so that the rate of enzyme productin responds to the physiologcal need of the cell done to achieve cellular economy’’

1. Allosteric Regulation. 2. Activation of Latent Enzyme. 3. Comprtmentation of Enzymes of different

Pathways.

4. Control of Enzyme Synthesis.

5. Enzyme Degradation.

REGULATION OF ENZYMES Control of metabolic pathway occour through

modification of Enzyme activity. One or more key enzyme on the pathway. Can be

involved in regulation. One enzyme that regulate is called rate limiting

enzyme or key enzyme of that path –way.Usually this is first enzyme.

This enzymes activity change in quantity of enzyme present orIntrinsic catalytic efficiency of enz. Molecule.

CHANGE IN ENZYME QUANTITY Absolute Quantity of Enz.is determined by balance

(Rate of Synthesis&Degradation) Enzyme whose synthesis is increased by inducer

are called inducible enz.Eg. Tryptophan pyrolase,Tyrosine Transaminase and HMG co-A Reductase.

Enz. Whose conc. Always maintained at particular level,independent of Inducor are called constitutive enz. Eg. Hexokinase.

Sometimes accumulation of metabolite inhibit its own synthesis.These are called repressor.

CHANGE IN CATALYTIC EFFICIENCY OF ENZYME Catalytic effeciency is regulated is modulated by A. Allosteric Regulation. B. Covalent modification. A. ALLOSTERIC REGULATION: Here the site is

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

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

A.Activator A.Inhibitor. Allosteric Activator. Hexokinase: ADP Isocit.Dehydr. ADP Glu.Dehy. ADP Pyruvate Carboxylase Acetyl CoA

Allosteric Inhibitor. Glucose-6-P,ATP Glucose-6-P,ATP ATP, NADH.

ADP

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

homotropic effect. HETEROTROPIC EFFECT: Effector molecule is a

substance other than substrate. SECOND MESSENGER:Binding of many

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

Hormone is first messenger. Cont……

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

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

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

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

CONFERMATIONAL CHANGES IN ALLOSTERIC ENZYMES.

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

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

EXAMPLE OF 2nd MESSENGER

GLYCOGEN BREAKDOWN.

GLYCOGEN SYNTHESIS.

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 ReceptorThe third group: Ion-channel-linked Receptor

Gilman, Rodbell (1994)

Glycogen breakdown

Glycogen

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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 INHIBITION

Enzyme is inhibited by end product of reaction.

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

converts A in to B.

COVALENT MODIFICATIONS Two well known processes A. PHOSPHORILATION. B. PARTIAL PROTEOLYSIS. A. Phosphorilation-dephosphorilation:many

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

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

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PARTIAL PROTEOLYSIS

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

This convertion takes place as a selective proteolysis.

It is ir-reversible process Pepsinogen to pepsin Trypsinogen to trypsin.

MICHAELIS CONSTANT (Km) It is defined as the conc. Of the

substrate at which the reaction velocity is half of the maximum velocity.

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

achieves maximal catalytic efficency at low substrate conc.

SIGNIFICANCE Glucokinase has high Km is low affinity for

glucose

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

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

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

INHIBITORS

Inhibitors Inhibitors are chemicals that reduce the rate of

enzymic reactions. The are usually specific and they work at low

concentrations. They block the enzyme but they do not usually

destroy it. Many drugs and poisons are inhibitors of enzymes

in the nervous system.


The effect of enzyme inhibition Irreversible inhibitors: Combine with the

functional groups of the amino acids in the active site, irreversibly

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


The effect of enzyme inhibition Reversible inhibitors: These can be

washed out of the solution of enzyme by dialysis.

There are two categories


The effect of enzyme inhibition2. Non-competitive: These are not influenced by the

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

Examples Cyanide combines with the Iron in the enzymes

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

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


Applications of inhibitors

Negative feedback: end point or end product inhibition

Poisons snake bite, plant alkaloids and nerve gases

Medicine antibiotics, sulphonamides, sedatives and stimulants


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

A B C D E F

Enzyme pathways

eFeDeCeAeB

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

This is possible because of enzyme specificity


End point inhibition

The first step (controlled by eA) is often controlled by the end product (F)

Therefore negative feedback is possible

A B C D E F

The end products are controlling their own rate of production

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

eFeDeCeA eB

Inhibition


ATP is the end point

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

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

is inhibited Respiration slows down and less ATP is

produced As ATP is used up the inhibition stops and

the reaction speeds up again


The switch: Allosteric inhibition Allosteric means “other site”

E

Active site

Allosteric site


Switching off

These enzymes have two receptor sites

One site fits the substrate like other enzymes

The other site fits an inhibitor molecule

Inhibitor fits into allosteric site

Substratecannot fit into the active site

Inhibitor molecule


The allosteric site the enzyme “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


A change in shape

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

The enzyme’s molecular shape changes The active site of the substrate changes The substrate cannot bind with the substrate


Negative feedback is achieved The reaction slows down This is not competitive inhibition but it is

reversible When the inhibitor concentration diminishes

the enzyme’s conformation changes back to its active form


Phosphofructokinase The respiration pathway accelerates and

ATP (the final product) builds up in the cell As the ATP increases, more and more ATP

fits into the allosteric site of the phosphofructokinase molecules

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

Respiration slows down


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-

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’


The effect of enzyme inhibition Irreversible inhibitors: Combine

with the functional groups of the amino acids in the active site, irreversibly.

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


The effect of enzyme inhibition Reversible inhibitors: These can be

washed out of the solution of enzyme by dialysis.

There are two categories.


The effect of enzyme inhibition1. Competitive:

These compete with the substrate molecules for the active site.

The inhibitor’s action is proportional to its concentration.

Resembles the substrate’s structure closely.

Enzyme inhibitor complex

Reversible

reaction

E + I EI


CLINICAL APPLICATIONS OF COMPETITVE INHIBITORSDRUG ENZYME TRUE SUB. Clinical App.

ALLOPURINOL

XANTHINE OXIDASE

HYPOXANTHENE

GOUT

SULFONAMIDE

Dihydro pteroate Synthase

PABA ANTIBIOTIC

ETHANOL Al.Dehy. METHANOL METHANOL POISONING

The effect of enzyme inhibition2. Non-competitive: These are not influenced by

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

Examples Cyanide combines with the Iron in the enzymes

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

groups.

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


Applications of inhibitors

Negative feedback: end point or end product inhibition

Poisons snake bite, plant alkaloids and nerve gases.

Medicine antibiotics, sulphonamides, sedatives and stimulants


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

DIAGNOSTIC SIGNIFICANCE ISOENZYMES: 1.CPK: MM,BB and MB.(Skeltol

muscles,Brain and Myocardium) 2. LDH:LDH-1 (H4) Myocardium LDH-2 (H3M) Erythrocytes. LDH-3 (H2M2) Brain. LDH-4 (HM3) Liver,Muscles. LDH-5 (M4) Liver, Muscles.

Plasma enzymes are of two types:Plasma 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. FUNCTIONAL: Lipoprotein FUNCTIONAL: Lipoprotein lipase,Pseudocholine estrase,blood lipase,Pseudocholine estrase,blood coagulation.coagulation.

3.3. NON FUNCTIONAL ENZYMES:NON FUNCTIONAL ENZYMES:

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

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

A. Plasma Enzymes as diagnostic 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 The level of these enzymes in plasma correlates with the extent of plasma correlates with the extent of tissue damage.tissue damage.

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 diagnostic to damage of a particular tissue; particular tissue; e.g. The enzyme alanine e.g. The enzyme alanine aminotransferase (ALT) is aminotransferase (ALT) is abundant in the liver and the abundant in the liver and the appearance of elevated levels of appearance of elevated levels of ALT in plasma indicates damage to ALT in plasma indicates damage to the liver.the liver.

Intracellular Distribution of Diagnostic Enzymes

LiverLiver HearHeartt

PancrePancreasas

SalivarSalivary y GlandsGlands

BonBonee

MusclMusclee

BiliarBiliary y TractTract

ProstaProstatete

LDLD55

ALTALTASTAST

LDLD11

ASTASTCKCK

LPSLPSAMSAMS

AMSAMS ALALPP

CKCK ALPALPGGTGGT

ACPACP

ISOENZYMES Isoenzymes or Isozymes are physically distinct

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

Useful for diagnosing diseases of different organs.

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

are produced by different genes.

IDENTIFICATION OF ISOZYMES 1.Agar gel or PAGE.They have different mobility.

2.Heat stability. 3.Inhibitors.Isozymes may be sensative to different

inhibitors.eg.tartrate labile. 4. Km value or substrate specificity. Eg.Glucokinase

has high Km and Hexokinase has low Km for Glucose. 5.Co-Factors.Eg Mitochondrial isocitrate

dehydrogenase is NAD dependent,Cytoplasmic NADP dependent.

6.Localisation:H4 heart,M4 Muscles. 7.Specific antibodies identify sp.Isozyme.

Isoenzymes

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

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

Diagnostic Significance Enzymes The levels of diagnostic

enzymes determine the amount of damage in tissues.

B. Isoenzymes and Heart Diseases 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 plasma level of certain isozymes of the enzyme Creatine kinase (CK) level is the enzyme Creatine kinase (CK) level is determined in the diagnosis of myocardial determined in the diagnosis of myocardial infarction.infarction.

DIAGNOSTIC IMPORTANCE Creatine

phosphokinase Alkaline phosphatase

CPK-BB,CPK-MM,CPK-MB

Alfa 1 ALP (liver) Alfa 2 ALP Heat labile

(Liver)& Heat stable (Placenta)

Prebeta ALP (Bones) Gama ALP (Colon) Regan ALP (Tumours)

DISORDERS DIAGNOSED BY ENZYMES1) 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

CARDIAC MARKERS

CPK (MB) LDH (1) CARDIAC TROPONIN (I)&(T) BRAIN NATRIURETIC PEPTIDE (Marker of Ventricular function) AST ALT

LIVER MARKERS

ALT (Alanine amino transferase) ALP (Alkaline phosphatase) NTP (Nucleotide phosphatase) GGT (Gama glutamyl Tranferase)

PROSTATE MAR

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

MUSCLE MARKER

CK (MM) AST (Aspartate Amino Transferase) ALD (Aldolase)

BONE MARKER

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:

AST LD1 CK

2. Hepatic Disorders

a)a) Hepatocellular DisordersHepatocellular Disorders:: (1) Viral hepatitis: Hepatitis B & Hepatitis (1) Viral hepatitis: Hepatitis B & Hepatitis

C.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. Skeletal Muscle Disorders

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. Bone 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. Acute Pancreatitis

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

LipaseLipase AMSAMS

6. Salivary Gland Inflammation: In Mumps:In Mumps:

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

7. Malignancies

a)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 in: phosphatase (ALP) increase 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.

B. Isoenzymes and Heart Diseases 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 plasma level of certain isozymes of the enzyme Creatine kinase (CK) level is the enzyme Creatine kinase (CK) level is determined in the diagnosis of myocardial determined in the diagnosis of myocardial infarction.infarction.

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

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

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

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

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

Alkaline Phosphatase

1.Alfa1-ALP Liver 2.Alfa2-ALP Liver (Heat Labile) 3.Pre Beta-ALP (BONES) 4.Gama ALP (Ulcerative Colitis) 5.Regan ALP (Bronchogenic cancer)

Sialic Acid Residues

ENZYMES IN OTHER BODY FLUIDS Adenosine deaminase in pleural

fluid :Elevated in Tuberculosis not in Malignant effusion.

LDH; In CSF,Pleural fluid & Ascitic Fluid. Elevated levels in Malignacy.

Enzymes as Therapeutic Agents Dissolving thrombus,Streptokinase,Urokinase.

Asparaginase used in some leukemias. Deoxyribonuclease is adminstered via respiratory route to

clear viscid secretions in pt. of cystic fibrosis. Serratiopeptidase is used to minimise edema in acute

inflamatory conditions. Hyaluronidase for hypovolumia Hemocoagulase used as hemostat. Fungal Diastase &Pepsin used as digestive enz. Ribozymes &Abzymes Streptodornase; DNA applied locally. Alpha-1-ant-trypsin; Emphysema.

ENZYMES USED FOR DIAGNOSIS Urease Urea. Uricase Uric Acid. Glucose Oxidase Glucose. Peroxidase Cholesterol. Hexokinase Glucose. Lipase Triglycerides. Alkaline phosphatase ELISA. Restriction endonuclease RFLP

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-

Enzyme Active Site Is Deeper than Ab Binding

Instead, active site on enzymealso recognizes substrate, butactually complementally fits the transition state and stabilized it.

Ag binding site on Ab binds to Agcomplementally, no further reactionoccurs.

X

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

HIV protease vs Aspartyl protease

Asymmetric monomer

↓ HIV protease HIV protease (homodimer)

HIV Protease inhibitor is used in treating AIDS

Symmetricdimer

Asp

subunit 2

↑Aspartyl protease (monomer)

subunit 1

Asp

domain 1 domain 2

Asp Asp


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

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)

SH2domain


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




Glycogen breakdown

Glycogen

Jua

ng

RH

(2

00

7)

BC

ba

sics

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


SH2domain


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




Glycogen breakdown

Glycogen

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ng

RH

(2

00

7)

BC

ba

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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


Modification of Subtilisin and Its Activity Change

No enzyme 1

Asn155 → Leu ● ● ● 10,000,000

(Asn155 stabilizes transition state)

His & Asp → Ala ● ○ ○ 37,000Ser, His & Asp → Ala ○ ○ ○ 4,000Subtilisin ● ● ● 10,000,000,000

Active Site RelativeModification Triad: Ser His Asp activity

Ser → Ala ○ ● ● 5,000Asp → Ala ● ● ○ 330,000

Serine Protease and AchEChymotrypsin – Gly – Asp – Ser – Gly – Gly – Pro – Leu – Trypsin – Gly – Asp – Ser – Gly – Gly – Pro – Val – Elastase – Gly – Asp – Ser – Gly – Gly – Pro – Leu –Thrombin – Gly – Asp – Ser – Gly – Gly – Pro – Phe –Plasmin – Gly – Asp – Ser – Gly – Gly – Pro – Leu –Acetylcholinesterase – Gly – Glu – Ser – Ala – Gly – Gly – Ala –

Chymotrypsin – Val – Thr – Ala – Ala – His – Cys – Gly – Trypsin – Val – Ser – Ala – Gly – His – Cys – Tyr – Elastase – Leu – Thr – Ala – Ala – His – Cys – Ile – Thrombin – Leu – Thr – Ala – Ala – His – Cys – Leu – Plasmin – Leu – Thr – Ala – Ala – His – Cys – Leu – Acetylcholinesterase – – – – – – – – – – – – – His – – – – – – – –

Ser

19

5

Chymotrypsin – Thr – Ile – Asn – Asn – Asp – Ile – Thr –Trypsin – Tyr – Leu – Asn – Asn – Asp – Ile – Met – Elastase – Ser – Lys – Gly – Asn – Asp – Ile – Ala – Thrombin – Asn – Leu – Asp – Arg – Asp – Ile – Ala – Plasmin – Phe – Thr – Arg – Lys – Asp – Ile – Ala – Acetylcholinesterase – – – – – – – – – – – – – – Asp – – – – – – –

His

57

Asp

102

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.244

H

AchE

AchE Has Similar Catalytic Mechanism

O -

C

H

O CH3

CH3–C–O–CH2–CH2–N–CH3

CH3

+

H-O-H

AchE

O

C

H

OCH3–C

CH3

HO–CH2–CH2–N–CH3

CH3

+

H2O

AchE

O -

C

H H

O CH3–C–OH

Adapted from Dressler & Potter (1991) Discovering Enzymes, p.243

AchE

O -

C

H

OCH3–C

CH3

O–CH2–CH2–N–CH3

H CH3

+

↓Deacylation

Acylation↑

Different Enzymes Might Adopt Same Mechanism

O -

C

Sesame Triad

Hi, Everybody!Hi, Everybody!

← Useful

↙ Amusing


Divergent evolution

Asp--His--Ser

Asp--His--Ser

Convergent evolution

Convergent and Divergent

TrypsinChymotrypsin Elastase Thrombin Plasmin

AcetylcholinAcetylcholinesteraseesteraseAcetylcholinAcetylcholinesteraseesterase

ThyroglobulinThyroglobulinThyroglobulinThyroglobulin

C NC

C

H

O

CC

CO

O

Ester bond

Peptide bond

EvolutionEvolutionMolecularMolecular

hydrolyzeacetylcholine

Serine Protease


Activity Regulation of Glycogen Phosphorylase

PA

PA

P

P

A

A

Covalent modificationCovalent modification

P

P

GP kinase

GP phosphatase 1

No

n-co

valent

No

n-co

valent

PA

PA

P

PPA

PAA

A

A

AMP

ATPGlc-6-PGlucoseCaffeine

GlucoseCaffeine

spontaneously

R

T

R

T

Ga

rre

tt &

Gris

ha

m (

19

99

) B

ioch

em

istr

y (2

e)

p.6

79

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

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

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(2

00

4)

BC

ba

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(199)enzyme 2011

Health & Medicine