2 Enzymes

ENZYMESENZYMES

Enzymes are the catalysts of biological systems

Most enzymes are proteins that accelerate chemical reactions by factors of at least million.They catalyze hundreds of stepwise reactions by which nutrient molecules are degraded, chemical Energy is conserved and transformed and biological macromolecules are made from simple Precursors .

Catalyst is a substance that accelerate the rate of a chemical reaction without it being changed duringThe process .

The substance that is being changed by the catalyst is known as the substrate.

IntroductionIntroduction

The study of enzymes is of practical importance :In many inheritable disorders, there may be a deficiency, absence or excessive activity of an enzyme. One can measure the activities of many enzymes in plasma or certain tissues.

Enzymes are used in industry for food processing .

Characteristics of EnzymesCharacteristics of Enzymes::

1 )Enzymes have immense catalytic power. They accelerate the rate of reactions by many million folds. Reactions that would normally will not occur

Under physiological PH and temp. conditions are induced by enzymes. For example the breakdown of sugars To carbondeoxyde and water.

2 )Enzymes are very specific to their substrates. This way the processes are very efficient and there is no loss of energy.

Trypsin cleaves on the Carboxyl side of lysineOr arginine only

Thrombin cleaves only Arg-Gly bonds.

DNA polymerase I synthesizes very accurately DNA strands . It can make only one mistake in a millionBecause it proofreads the product during synthesis and corrects its mistakes .

3 )The catalytic activities of many enzymes are regulated

A) Feedback inhibition: An enzyme that catalyzes the first step in a biosynthetic pathway is inhibited by the end product. When the concentration of the end product is high enough it can bind directly to the enzyme and

inhibit its activity. When the level of the end products drops, it will be released and allow the enzyme to function.

B) Regulatory proteins: Proteins can bind to enzymes and regulate their activities. The activities of many enzymes is regulated by a small protein Calmodulin. Calmodulin is a sensor of Ca2+ in the cell. The binding of Ca2 + to multiple sites in calmodulin induces a conformational change that converts it to an active state. It can then

bind many enzymes and modify their activities .

Calmodulin )no calcium)Calmodulin )with calcium)

Myosin light Chain kinase

C) Covalant modifications: Many enzymes are controlled by the reversible attachment of phosphoryl groups to specific residues. Kinases catalyze the attachment of phosphoryl groups. Phosphatases catalyze their

removal by hydrolysis.

D) Proteolytic activation: Some enzymes are synthesized as inactive precursors and are activated in the appropriate time and place by proteolytic cleavage. For example trypsinogen is synthesized in the pancreas and is activated

by proteolytic cleavage in the small intestine to form an active trypsin. This mechanism is unidirectional . In happens also in blood clotting. The inactive precursor is called Zymogene.

4 )Enzymes transform different forms of energy: *Light energy is converted into chemical bond energy )photosynthesis)* In mitochondria; sugar energy is changed to chemical bond energy of

ATP.* The energy of ATP is converted in muscle to mechanical energy.

5 )Enzymes cannot alter reaction equilibria:

A B [B ]

[A ] =100

k1

k2k= velocity constant )mol/sec)

k1=10-4 k2=10-6 No enzyme:

With enzyme: k1=104 k2=102

Therefore enzymes accelerate the attainment of equilibria butdo not change it .

6 )Active sites of enzymes have some common features:

A. The active site is relatively a small part of the total protein.

B. The active site is a three dimensional entity. It is composed from amino acids that are far apart in the sequence.

Lysozyme: the catalytically important residuesAre in red and green. Other residues that contributeBinding of the substrate are in yellow .

C. Substrates are bound to enzymes by multiple weak interactions: Many electrostatic bonds, hydrogen bonds , hydrophobic bonds and Van Der Waals forces. The enzyme and the substrate have complementary shapes.

D) Binding sites are clefts. Great specificity. Only molecules that contribute to catalysis will fit in the cleft .

Two theories to explain the specificities: 1) the lock and key theory by Emil Fhisher )1894)

The active site of the unbound enzyme is complementary

in shape to that of the Substrate.

2 )The theory of induced fit by Daniel Koshland )1958).

The enzyme and the substrate change shapes upon binding to

Each other. The active site is Complementary to that of the Substrate only after binding.

7 )Formation of an enzyme-substrate complex is the first step in catalysis .

The first step is the formation of

ES complex

The main proof is experimental:At a constant concentration ofEnzyme, the reaction rate increaseWith increasing of substrate Concentration until a maximal Velocity is reached

In x ray crystallography or electron microscopy, one can “freeze” the complexes.

8 )Enzymes accelerate the reaction rates by lowering the activation energies.

Energy changes during the reaction: The free energy of the system is plotted against the progress of the reaction .

Ground states: The starting point for either forward or reverse reaction.The equilibrium between S and P reflects the difference in the free energies

of their ground states. If P is lower than S, ∆G is negative and the equilibrium Favors P )the concentration of P in equilibrium is higher than S.)

The rate of the reaction is dependent on energy barrier that is required For the alignment of reacting groups, formation of transient unstable charges Bond rearrangements that occurs in the transition state .

No Enzyme

EnzymeTransition state: The top of the energy hill at which decay to S or P states isEqually probable. The transition state is not a chemical species )like ES or EP).It is a thermodynamic description of the reaction.

The difference between the energy levels of the ground state and the transition states is called activation energy.

The higher the activation energy, the slower the reaction. The rate of a reaction can be increased by raising theTemperature, therefore increasing the number of molecules with sufficient energy to overcome the barrier.

Alternatively, the activation energy can be lowered by a catalyst .Therefore, enzymes increase the rate of reactions by lowering the activation states of the reactions

Many reactions have several steps involving the formation and decay of transient chemical species called reaction intermediates. The ES and EP

Complexes are intermediates. They occupy valleys in the reaction diagram.In a multistep reaction the overall rate is determined by the step with the

highest activation energy-the rate limiting step.

How an enzyme lowers the activation energy of a reaction??

Lets imagine a metal stick that shouldBe broken. The energy to break it is Represented by ∆G

The enzyme is represented by aMagnet that binds the stick. A bad

enzyme will complement the substrateIn its initial condition. It will not be ableBreak the stick. In order to do so, it willFirst to break the existing magnetic contactsAnd create new one. This “enzyme” will slowThe reaction rate.

The magnet weakly binds the stick. The Magnet and stick fully complement each Other in the transition state when the stickIs almost broken. The magnetic forces include Parts of the stick different from the point of breakage.These interactions provide the energy needed to break The stick. Therefore they lower the energy need to break The stick.

In real terms: The multiple weak interactions between the enzyme and the substrate in the transition state lower the energy needed for the reaction to take place .

Binding of substrates brings reactants to proximity

Binding of substrates orients the substrates and allowsthe active chemical groups to react .

Hexokinase:

Glucose+ATP Glucose 6-phosphate

Hexokinase binds glucose and ATPIn proximity and in the right Conformation to allow the Reaction to occur .

Enzyme kinetics-the Mechaelis Menten model

The plot describes the rate of reaction in the presence of constant Amount of enzyme and growing amounts of substrate. Shows that In high substrate concentrations, the reaction gets to a value of

maximum velocity. How can this behavior be represented mathematically? It can be explained by a stage of a complex formation

Of the enzyme with its substrate )one enzyme, one substrate) :

Assumptions: 1) At enough early stages of reaction, product Concentration is 0. We can assume that there is no reverse Reaction between E and P.

2 )The formation of the ES complex is the faster Stage. The Catalytic stage is the slower rate limiting stage.Therefore the velocity of the reaction depends on the Concentration of ES and a catalytic constant )k2).

V=k2]ES[ The maximal velocity of a reaction )Vmax) occurs when All the enzyme is in a complex with the substrate:

Vmax=k2Et

[ES ]is not a measurable concentration. We can Measure substrate and the total concentrationsOf the enzyme. Therefore, to find the V of a Reaction, we have to have a formula that use these concentrations .

[E]t =]E[+]ES[

Total enzyme

Freeenzyme

Enzyme inES complex

The reaction starts by mixing enzymes and substrates.The ES concentration builds up but quickly reaches A steady state which remains almost constant.

At steady state conditionsThe formation of ES complex Equals the rate of breakdown Of the ES complex

d]ES[ =0 dt

If all the constants are put in One side of the equation, we canCombine all of them to oneConstant, Michaelis constant Km.

In such case: ]ES[= ]E[ ]S[ Km

If we put in the equation ]E[=]Et[ +]ES[ we get: ]ES[=]Et[ ]S[ –]ES[ ]S[ Km Km

This regenerates to: ]ES[=]Et[ ]S[ [ S+]Km

If we insert the value of ]ES[ in the equation for velocity V=k2]ES[ We get V= k2 ]Et [ ]S[ [ S+]Km

When all the enzyme is occupied in The reaction, then V=Vmax

Therefore, we can express MichaelisMenten equation this way :

What could be studied from Michaelis Menten equation ?

1 )Lets assume that V is measured in very high substrate concentrations: Km<<]S[

In this case we can neglect Km and V=Vmax]S[ Km+]S[

And V=Vmax

Therefore at these conditions the velocity of the reaction is not dependent at the concentration of the substrate.

2 )In very low concentrations of substrate ]S[ <<Km then we can neglect S and V= Vmax ]S[ KmIn small concentrations of S the reaction rate is directly dependent on ]S[ )first order reaction).

3 )When Vint = Vmax 2

Vmax=Vmax]S[ 2 Km+]S[

Then: [S=]Km The reaction reaches to its half maximal velocity when the concentration of the substrate

equals Km . Km is equal to the substrate concentration at which

the reaction velocity has attained half of its maximum value. Thus, Km is a measure of the substrate

concentration required for effective catalysis to occur .

The significance of Km, kcat )k2) and kcat/Km

Km is often associated with the affinity of enzyme with the substrate. This is true when k cat is a relative small value

Km= k-1 + kcat

k1

However, a high value of kcat will affect a high value of Km and in this case Km willNot reflect the affinity .

kcat Gives a direct measure of the catalytic production of product under optimum conditions )saturated enzyme).

The unit is sec-1-known also as the turnover number: the number of substrate molecules turned over per Enzyme molecule per second.

The ratio kcat / KM is convenient measure of enzyme efficiency. We can understand this relation better if we considerA situation of very low substrate concentration. ]S[<<KM . In such case most of the enzyme is free ]E[t~]E[.

The equation becomes: V=kcat ]E[]S[ KM

kcat / KM behaves as a second order rate constant for a reaction between substrate and free enzyme .This ratio provides a direct measure for enzyme efficiency and specificity .

The value of kcat/ KM can giveA measure for the efficiency And specificity of enzymes.

Let us test whether a enzymatic reactionBehaves according to Michaelis Menten.And to measure values for kcat and KM.

One can mix an enzyme and a substrateAnd follow the disappearance of substrate With time. The velocity will change with Time and it is very difficult to follow.

It is easier to set up reactions with one Concentration of enzyme and several Concentrations of substrate. We know The initial substrate concentration. The Change in ]S[ is linear if measured for A short time. Therefore we can get values For V for different ]S[ .

We can now get the values for Km and Vmax by changing the Michaelis Menten equation to get

A linear graph .

The double reciprocal graph isKnown as the Lineweaver BurkEquation:

When ]S[ ∞= Then 1=0[ S]

The intercept of the graph with the coordinate of 1/V will give the value

Of VmaxWhen we know Vmax and ]E[t we can calculate the Value of kcat.. Because Vmax= kcat ]E[t

When V=∞ then 1/V=0. In this situation 1[/S-=]1/KM.Therefore the intercept of the graph

With the coordinate of 1/]S[ will give the value ofKM.

Enzyme Inhibition

Many different kind of molecules inhibit enzymes by a variety of ways .A major distinction made between reversible inhibition and irreversible inhibition.

The reversible inhibitor involves non covalent binding of the inhibitor and can always be reversed.The irreversible inhibitor is bound covalently to the enzyme . The action of toxins and poisons is usually The action of irreversible inhibitors that kill by permanently incapacitating key enzymes.

Reversible inhibitionThere are different modes of inhibitions that differ in the mechanism by which they decrease enzymes activity:

Competitive inhibition:

A molecule resembles the substrate that can compete with the substrate on binding to the active site. The moleculeCannot undergo the catalytic step and therefore called an competitive inhibitor .

Both substrate and inhibitor can fit in The active site. Substrate can be processedBy the enzyme, but inhibitor cannot.

The I binds the enzyme and therefore there is a practical lower Concentration of E to bind S. Consequently there is less ES complex.Increasing the concentration of ]I[ will cause an increase in the Concentration of EI . Thus we expect that the enzyme wouldAct as if its KM was increased by the presence of the inhibitor .

In the presence of a competitive inhibitor one needs higher substrate Concentrations to get to Vmax.

[S ]

V

-I +I

KM KMapp

Vmax

A competitive inhibitor changes the apparent KM

But does not affect Vmax.

Noncompetitive inhibition

When a molecule can bind to a second site on the enzyme surface in such a way that it modifies k cat. It can for example distort the enzyme so the catalytic process is not efficient )Allosteric effect). The inhibitor is this case does not

resemble the substrate. The simplest case to consider is one in which the inhibitor does not interfere in any way with the substrate binding but completely prevents the catalytic step .

The result is that the KM is not influenced By the inhibitor. However the inhibitor Affects the apparent kcat . The apparentKcat .decreases with increasing I. The change Is actually the lower concentration of ES Complex that is available for the catalyticProcess. Therefore Vmax is affected .

KM

The apparent Vmax depends at the concentrationOf I: Vmax decreases as the concentration of I increases.

In reality the situation can be more complex .For example if the complex ESI can undergo The catalytic process slowly or the binding ofThe inhibitor affect both KM and kcat . The latter Case is called mixed inhibition .

Uncompetitive inhibition

The inhibitor can bind only to the ES complex .

As is the other cases the complex of ESI cannot Undergo the catalytic stage .

In this inhibition both the KM and kcat are affected.

[S ]

V

-I

+I

KMKMapp

Vmax

Vmaxapp

In this case Vmax is affected as in in noncompetitive inhibition,Because there is less ES complex available for catalysis.Surprisingly the apparent KM decreases with increasing Concentration of the inhibitor. The reason is that ES can becomeESI. To reach equilibrium, more E+S is used to make more ES.

The overall effect is inhibition of the reaction because At any substrate concentration there is less ES Complex that is available for catalysis .

Irreversible Inhibition

Some chemical substances can covalentlybind to the active site of enzymes and Block the binding of the substrate.

Some of those are natural toxins and someMan-made toxins.

Diisopropyl fluorophosphate )DFP) React with the serine group in In the active sites of serine proteases .

Many of these inhibitors are similar to the tetrahedral transmission state of enzymes with their substrates and thereforeBind with great affinity .

Sarin that is known as Nerve gas binds to serineIn the active site of AcetylCholinesterase and blocks Synaptic transmission.

TPCK is an inhibitor ofChemotrypsin. The phenylGroup fits into the active Site and position the Cl To react with imidasole Group of His 57.

Penicillin inhibits Glycopeptide transpeptidaseThat forms cross links in theBacterial cell wall. CovalentlyBinds to Ser in active site.

Enzyme activity is affected by pH

Enzymes have an optimum pH range for their activity .

Amino acid side chain in the active site may act as weak acids and bases .It is critical for the catalytic function to maintain state of ionization .

Ionic interactions are also critical to maintain the right structure ofThe enzyme .

The optimal pH range of an enzyme helps sometime to know what amino Acid is involved. A change in the activity around pH 7 suggest that His is involved .

Pepsin which Hydrolyzes peptideBonds in the stomachHas an optimum ofAbout 1.6.

Glucose 6 phosphataseFunctions in the liverHas an optimum of

7.8 ,responsible To release glucose To the blood.

Examples for Enzymatic mechanisms of catalysis

The serine proteases- Chemotrypsin

We will discuss a family of enzymes that illustrate some general Principles: Binding site, enzyme-substrate complex, transition states,Catalysis .

Serine proteases function to hydrolyze peptide bonds” Trypsin Chemotrypsin, thrombin .Chemotrypsin is specific to peptide bonds adjacent to aromatic amino acids .

Trypsin is specific to peptide bonds adjacent to basic amino acids.

The specificity isAchieved by the Structure of the Pocket. In the caseOf trypsin thereIs a negativelyCharged amino acidAt the pocket.

In Chemotrypsin The pocket contains Hydrophobic aminoAcids.

The three dimensional structure of all serine proteases is very similar; evolutionary conserved .

The catalytic domain in this group is composed of three critical amino acids: Ser, His, Asp .

The catalytic triad: In the Absence of substrate His 57Is protonated. With the addition Of substrate a proton is Transferred from Ser 195 toHis 57 . The positively charged Imidazole ring is stabilized by Electrostatic interaction withNegatively charged Asp 102.

Chemotrypsin is composed of three polypeptides linked by disulfide bonds. The active site amino acid residuesAre grouped together in the three dimensional structure .

Steps in the hydrolytic cleavage of a peptide bond by chemotrypsin.

Hexokinase Glucose and ATP to form Glucose 6-phosphate.

The active site can bind also water instead of glucose )OH of water resembles OH of C6), yet the enzyme discriminatesbetween glucose and water and prefers glucose by factor of 106.When glucose )and not water) binds it induces a conformational change in the enzyme- induced fit. The binding

energy derived from the interaction induces a conformational change that allows the formation of the active catalytic site

The conclusion of a conformational change was supported by studies with xylose:

It is similar enough to glucose to bind, but cannot be phosphorylatedIts binding induces a conformational change, and therefore the enzymeIs “tricked” to phosphorylate water .

In the case of hexokinase the specificity is not observed on the formation of ES complex but at relative rates of subsequent Catalytic steps.

Enolase: In glycolysis, reversible dehydration of 2-phosphoglycerate to phosphoenolpyruvate .

The two step reaction demonstrates a metal ion catalysis and provides an example of general acid-base catalysis And transition state stabilization .

Two Mg ions coordinate the binding of 2-

Phosphoglycerate to the Enzyme. Residue Lys actsAs a general base catalystReceiving a proton fromC2 .

Glu acts as a general Acid catalyst, donoringA proton to the –OH Leaving group The Mg ions making the C2 of phosphoglycerate which

Not very acidic, more acidic )lowering its pK) .

Regulatory strategies of enzymes:

Enzymes has to be catalytically active in the right time and in the right place.Enzymatic activity is regulated in four principle ways:

1 )Allosteric interactions. The activity of many multiple subunit enzymes is regulated by the binding of substrates,

Inhibitors, or activators that induce conformational changes . Later we will discuss the example of aspartate

Transcarbamoylase, an enzyme that is part of the biosynthesis of pyrimidines .

2 )Stimulation and inhibition by control proteins: Calmodulin that Senses the intracellular concentration of Ca2+ and activates many

Enzymes in the cell when Ca2+ levels rise.

3 )Reversal covalent modifications: Phosphorylation of certain amino acids in enzymes that is catalyzed by protein kinases. The phosphorylation normally induces conformational changes in the enzymes that will Either activate or repress their activities. The removal of phosphoryl groups by hydrolysis is catalyzed by

Protein phosphatases .

4 )Proteolytic activation: An irreversible conversion of an inactive to an active enzyme. Proteolytic cleavage of a peptide from the precursor

that is known as zymogene can expose the active site. Digestive enzymes like chemotrypsin, trypsin and pepsin and also

Blood clotting enzymes belong to this group .

Allosteric Enzymes

Enzymes that have several subunits and active sites and do not obey Michaelis Menten kinetics :

Allosteric enzymesAre represented by Sigmoidal dependenceOf reaction velocity onSubstrate concentration.

In allosteric enzymes the binding of a substrate to one Active site can affect the proparties of other active sitesIn the same emzyme molecule.

Cooperativity in substrate binding-Homoallostery.

Regulation of activity by other effector molecules-Heteroallostery.

At low substrate Concentration the Enzyme has low affinityTo substrate )high Km)At higher substrate Concentration the enzyme

has high affinity )lowKm.)

The weak binding conformation isT state, and the strong binding stateIs R state .

Why this behavior is needed ??

In extreme cases enzyme canRegulate a substrate level toConstant values. A substrate That is produced all the time.It accumulates to a critical level

[S]c . The enzyme becomes Extremely efficient at this Substrate concentration .This behavior helps to keep Homeostasis of dynamic systems.

Allosteric= other shape )enzymes that can adopt different shapes)

Heteroallostery

Heteroallosteric effectors can be inhibitors or activators.. They bind to remoteSites )not the active site). If the enzyme can exist in two conformational states

)T and R )that differ in the strength of substrate binding or in the catalytic rate.Binding of an activator stabilize the R state and shifts the equilibrium T R Towards R. Binding of an inhibitor to the T state stabilizes it and shift the Equilibrium towards T.

Two models to explain allosteric interactions

The sequential model and the concerted model.

Consider an allosteric enzyme of two subunits each contain a binding site.The T form has low affinity and the R form has high affinity .

The sequential model: The binding of substrate to one subunit induces a T R transition in that subunit and not in the other. The affinity of the other is increased Because the subunit interface has altered by the first substrate molecule .

The concerted model: The binding of substrate to one subunit will switch both from T to R State. Symmetry is conserved in this model and not in the sequential model.

Aspartate transcarbamoylase

Involved in pyrimidine biosynthesis. It is the first enzyme in the synthesis.

CTP

The enzyme is inhibitedBy the final product CTP

)feedback inhibition .)On the Other hand ATP stimulates the Enzyme. Both molecules don’t Affect Vmax, but only Km. ATP And CTP compete on binding to The same site on a regulatory Subunit .

The binding of carbamoyl Phosphate and aspartate to the enzyme is cooperativeAs can be seen by the sygmoidal Binding curve .

ATCase consists of separate catalytic and regulatory subunits:

The regulatory activity of the enzyme vanish when it is treated with a mercurial that reacts with the sulfhydryl groups. This treatment

Prevents the effect of ATP and CTP and the binding of substrate becomes Not cooperative. However, the maximal catalytic activity is not changed .

What happens ??Ultracentrifugation showed that the mercurials treatment dissociate ATCase Into two subunits. The sedimentationCoefficient of the native enzymeIs 11.6S whereas that of the Dissociated subunits is 2.8And 5.8S. Therefore the twoTypes of subunits can be separated And the mercurybenzoate groups can be Removed by adding mercaptoethanol.The large subunits are the catalytic, and are not affetced from CTP or ATP .The small subunits do not have catalytic activity and bind CTP and ATP-regulatory.The catalytic subunit consists of three c chains and the regulatory subunit from

2 r chains. When mixed one gets the full enzyme:

The ATCase is composed of district catalytic and regulatory subunitsThat interact in the native enzyme to produce its allosteric behavior.

The enzyme three dimensional structure: It was solved by x ray crystallography alone and as a complex with itsInhibitor CTP and substrates analogs. The enzyme contains a large central cavity

The regulatory subunits are found in the periphery consistOf two domains. The outer one contains a site for CTP and the inner oneInteracts with the C chain.Each R chain contacts two C chains and each C chain contacts

Two R chains .

The inner domain of the R chain contains a Zn ion That plays a role in Coordinating sulfur atoms Of four cysteine residues.

The catalytic site is found inThe center of the catalyticSubunit. Each enzyme Contains 6 catalytic sites .

The catalytic site: Carbamoyl phosphate binds first by multiple hydrogen and electrostatic interactions. Then aspartate binds and its amino group attacks the carbonyl carbon atom of carbamoyl phosphate.

A tetrahedral transition State in the catalysis:

PALA is a potent inhibitor That resembles the two substrates Complex and the transitionState.PALA was most important for the understanding

Of catalysis by the enzyme. It binds with High affinity to the six catalytic sites. Because of the Negative charge of PALA it binds to four arginines and one lysine in the active site. It also binds with many hydrogen Bonds. The active site is formed from residues belonging to two catalytic chains )Ser 80 and 84 from one chain, the

Rest are from a second chain .).

Histidine 134 stabilize the transition state: When the amino group of aspartate attacks the carbonyl group of carbamoylPhosphate, the oxygen becomes negatively charged. The protonated form of His 134 )positively charged) stabilize the Negative charge .

The binding of substrates )or PALA) induce large changes in the structure of ATCase:

Binding of PALA to the enzyme causesbig rotation of the catalytic and regulatorysubunits around two axis. All the catalyticsites are affected .

The movement of 240s loop )residues 230-245)Upon the binding of one substrate liberate Several side chains to interact with substratesAnd promote catalysis .

Which allosteric model )concerted or sequential) fits the properties of ATCase?

The sequential model predicts that the fraction of catalytic chains in the R state )fR) is equal to the fraction containingBound substrate )Y) .

The concerted model predicts that fR increases more rapidly than Y .

Succinate is an unreactive analog of aspartate. Y was determinedFrom the change in absorbance at 280 nm, and the value of fR from theSedimentation coefficient .

The change in fR leads the change in Y on addition of succinate, as predictedBy the concerted model .

Binding of nitromethane to tyrosine in the active group forms nitrotyrosine group that is colored )absorbs at 430nm).An essential lysine in the active Group was modified to block Binding of the substrate .Formed an hybrid enzyme.Catalytic labeled trimersWere mixed with native trimersThat can bind the substrate .Adding of succinate changed the Absorption spectrum of nitrotyrosineThus, binding to one trimer changedThe structure of the other trimer .

Allosteric activator shifts the conformational Equilibrium of all subunits to the R state, whereasInhibitor shifts it towards the T state .Normal regulatory subunits were mixed with Nitrotyrosine containing catalytic subunits.Addition of ATP in the absence of substrate increasedThe absorbance at 430 nm )like succinate). Thus ATP shifted the equilibrium to the R state .CTP decreased the absorbance at 430 nm thus this inhibitor Shifted the equilibrium to the T state .

Phosphorylation is an effective mean to switch the activity of enzymes

Kinases are enzymes that transfer the gamma phosphoryl group of ATP to serine,

threonine and tyrosine residues.

Phosphatases reverse the effect ofKinases by catalyzing the hydrolysis Of phosphoryl groups attached toThe proteins .

How the phosphoryl group change the activities of proteins ?

1 )It adds two negative charges to the protein. Can affect interactions with other protreins and can induce conformational changes.

2 )The phosphoryl group can form three hydrogen bonds.

Cyclic AMP activates protein kinase A by releasing inhibitory regulatorty subunits

Many hormones trigger the formation of cyclic AMP from ATP. Cyclic AMPServes as a messenger in the cell. Its main effect is the activation of protein Kinase A )PKA). PKA phosphorylates many enzymes on serine and threonineResidues .

The enzymes consists of two regulatory )R) subunits and two catalytic )C) subunits.Binding of two molecules of cAMP to each R subunit leads to dissociation of the

C from the R subunits. And the active site is freed .

Each R chain contains the sequence Arg-Arg-Gly-Ala-Ile that is a consensus siteFor phosphorylation except the presence of Alanine instead of serine. This peptideBlocks the active site from entry of substrate. cAMP releases this pseudosubstrateFrom the catralytic site.

Many enzymes are activated by specific proteolytic cleavage.

Proteolytic activation of is another mechanism to activate proteins. Inactive precursors )zymogens) are activatedBy specific cleavage of one or more peptide bonds .This modification occurs once in the life of an enzyme: it is not reversible.It can happen also outside cells because it dose not require ATP as phosphorylation of proteins requires .Biological systems that use proteolytic cleavage are:

1 )Digestive enzymes in the stomach and pancreas

2 )Blood clotting: ensures rapid and amplified response to trauma.

3 )Some protein hormones like insulin that is synthesized as proinsulin. Insulin is derived by proteolytic removal Of a peptide.

4 )Fibrous proteins collagen, the major constituent of skin and bones is derived from procollagen, a soluble precursor.

5 )Developmental processes: The metamorphosis of tadpole into a frog. Large amounts of procollagen are reabsorbed From the tail and the conversion to the active collagen occurs in a timely and precise mechanism .

Chemotrypsinogen is activated by a specific cleavage of a single peptide bond:

Chemotrypsin is a digestive enzyme. It hydrolyzes proteins in the small intestine. The precursor is synthesized in the

pancreas and stored inside membrane-bound granules .An hormonal stimulation cause the release of the content Into the intestine.

Chemotrrypsinogen is a single polypeptide consist of 245 residues with No enzymatic activity. Cleavage of peptide bond between arginine 15 And isoleucine 16 by trypsin activates the enzyme: π chemotrypsin.It can then act on other molecules and two di peptides are removed toYield α chemotrypsin- a stable form of the enzyme .The three resulting chains are linked by disulfide bonds .

How a single peptide cleavage turn chemotrypsinogen to an active enzyme?

The three dimensional structure revealed the key Conformational changes .

The new amino group of IsoLeucine 16 turns intoThe anterior of the molecule and interacts with Aspartate 194. Protonation of the amino group stabilizes

The active form the enzyme as seems by the dependenceOn enzyme activity on pH.

This interaction triggers a number of conformationalChanges that create the active site of the enzyme.

Pepsinogen cleaves itself in an acidic environment to form the active pepsin:

Pepsin digest proteins in the acidic environment of the stomach. Its pH optimum is 2 and it contains two aspartate

residues in the catalytic center. The precursor contains an Amino terminal segment that is proteolytically removed. TheActivation occurs spontaneously below pH 5. The rate of Cleavage is independent of the concentration of pepsinogenSuggesting that it is intramolecular cleavage .The precursor segment is very basic while pepsin is very acidic.The active site is blocked at neutral pH by the segment.Six lysine and arginine side chain of the segment form salt Bridges with side chains glutamate and aspartate of the Pepsin. Most importantly, a lys residue of the side chain Interactes with a pair of aspartates at the active site.When the pH is lower, these interactions are broken Because of protonation of the carboxylates. This exposes the Active site that hydrolyzes the peptide bond between the Precursor and the pepsin moieties .

Pancreatic trypsin inhibitor binds tightly to the active site of trypsin:

Because the activation step is irreversible, a different mechanism is needed to stop proteolysis .Specific protease inhibitors, like pancreatic trypsin inhibitor bind very tightly to the active site of trypsin -

One of the highest affinities in nature )0.1 pM) .

The inhibitor is very effective substrate analog. Lysine 15 Of the inhibitor binds to the Asp of the active site .

There are many hydrogen bonds between the twoChains creating an anti parallel β pleated sheet .The tetrahedral transition state of the active Ser residue With the inhibitor happens. The enzyme causes a very Slow cleavage between lys 15 and Ala 16 of the inhibitor .

The half life of this complex is several months !!!

Insufficient α-antitrypsin activity cause emphysema:

Antitrypsin is an plasmatic inhibitor of elastase. Elastase is a product of Neutrophils )white blood cells) . Like pancreaticTrypsin inhibitor, α antitrypson binds almost irreversibly to the active site of elastase. This inhibitor is of extreme

importance. A lys )53) for Glu mutation slows the secretion of this inhibitor from liver cells. People carrying Homozygous mutation have a disorder called emphysema. Elestase destroys alveolar walls in the lung by digesting Elastic fibers. People with emphysema breath much harder than normal .Cigarette smoking increase the chances of Heterozygotes to develop emphysema.

The smoke oxidizes Met 358 of the inhibitor .This methionine is an essential residue for binding elastase .

Activation by cleavage- Blood clotting

A cascade of enzymes: The active form of one clotting factor catalyze the activationOf the next. The many steps yield a large amplification, assuring a rapid response to

Trauma .

Two pathways: the intrinsic pathway is activated from non physiological surface likeGlass that cause clotting .

The Extrinsic pathway is activated by substances that are released from tissues as a consequence of trauma .

The final steps of both are common as they cause proper blood clotting .

The best characterized part of the process is the conversion of fibrinogenInto fibrin by thrombin.

Fibrinogen is made up by three globular units connected by two rods:

340 Kd protein consists of three kinds of Chains .

Thrombin cleaves four arg-gly peptide bondsIn the central globular region to release an A peptide from two α subunits and a B peptide from two β subunits .The fibrinogen molecule is called fibrin monomer.

Fibrin monomers spontaneously assemble into ordered fibrous Arrays called fibrin. Electron microscope shows that fibrin has A periodic structure every 23 nm.

Because fibrinogen is about 46 nm long it seems that monomers Associate to form half staggered array.

Why do fibrin monomers aggregate form ?

The peptides that are released carry highly negative charges that Keeps fibrinogen molecules apart. The release of these peptides byThrombin change the charges of the central globular region to Be more positive and the extreme globular regions that carry a Negative charge can interact with the central region. The newly Formed clot is stabilized of amid bonds between side chains of Glutamine and side chains of lysines in different monomer units.This cross linking binding is catalyzed by transglutaminase

)Factor XIII.)

Thrombin has a mass of 34 Kd and consists of two chains. The B chain is Similar to trypsin, chemotrypsin and elastase )belong to the serine proteases) .It is synthesized as a zimogen called prothrombin. Cleavage between Arg 274-

Thr 275 release 32 Kd fragment from the zymogen. Cleavage of Arg 323-Ile 324Yields the active thrombin. Like in chemotrypsin, an ion pair between the positivelyCharged amino group of Ile and a negatively charged group form the active site of

Thrombin .

Vitamin K is needed for normal clotting.Antagonists of vitamin K like DicoumarolAnd Warfarin prevent thromboses and Used clinically. Dicoumarol treated Animals synthesize abnormal prothrombinThat does not bind Ca2+. The capacity to bind Ca resides in the amino terminus region of prothrombin .

Normal prothrombin contains γ carboxyglutamate . The abnormal prothrombin formed after the administrationOf dicoumarol lacks this modified amino acid )contains glutamate) .All the first 10 glutamates in the amino terminus are carboxylated by a vitamin K-dependent mechanism .Carboxylation adds extra negative charge to glutamate and allows the chelation of Ca2+

The binding to Ca2+ anchors prothrombin to the plasma membrane where factor X )a serine protease and factor V ) a stimulatory factor) cleave and activate it. Once the amino terminal peptide that contains

The Ca2+ binding site is released, the active thrombin is release from the membrane and canActivate fibrinogen in the plasma .

Hemophilia:Genetically transmitted as a sex linked recessive trait. Heterozugous females are asymptomatic .Factor VIII is missing or reduced. Factor VIII is not a protease. It stimulates the activation of

X by factor IX .

Therapy: In the past , hemophiliacs were treated by transfusions of concentrated plasma fraction containingVIII. This carried risks of infection )HIV, HBV).The gene was cloned . The human gene of 186 Kb was transfected to the genome of Hamster cells and large Amounts of the protein were purified.The cloning of the gene helps also in prenatal screening for hemophilia mutations .

Fibrin Clots are lyzed by plasmin: Plasmin is a serine protease that hydrolizes peptide bonds in the triple stranded Connector rode regions of fibrin. Plasmin is formed by proteolytic activation of plasminogen, the inactive precursor.The conversion is carried by plasminogen activator )TPA). TPA has several domains .

The kringle domains bind TPA to fibrin clots Where it activates adhering plasminogen .The gene for TPA has been cloned and was expressed in

In cultured cells .

Clinical studies have shown that TPA administered intravenously Within an hour of the formation of a blood clot Markedly increases the likelihood of surviving a heart attack .

Dissolution of blood clots in vessels of the heart