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Enzymes are the catalysts of biological systems

Catalystis a substance that accelerate the rate of a chemical reaction without it being changed during
.The process

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

.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

: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 Enzymes
.Enzymes have immense catalytic power )1
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

)2Enzymes 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 lysine
Or arginine only

Thrombin cleaves only

.Arg-Gly bonds

DNA polymerase I synthesizes very accurately DNA strands . It can make only one mistake in a million
.Because it proofreads the product during synthesis and corrects its mistakes
)3The 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

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

Myosin light
Chain kinase

)Calmodulin )with calcium )Calmodulin )no calcium

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

)4Enzymes 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*
.The energy of ATP is converted in muscle to mechanical energy*
)5:Enzymes cannot alter reaction equilibria

k1 ]B[
A B )k= velocity constant )mol/sec 100 =
k2 ]A[

:No enzyme k1=10 -4 k2=10 -6 Therefore enzymes accelerate the attainment of equilibria but
:With enzyme k1=10 4 k2=10 2 .do not change it

: )6Active 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 residues

Are in red and green. Other residues that contribute
.Binding 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

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

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

The first step is

the formation of :The main proof is experimental
ES complex At a constant concentration of
Enzyme, the reaction rate increase
With 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 No Enzyme
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
Transition state: The top of the energy hill at which decay to S or P states is Enzyme
.)Equally 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 the
.Temperature, 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 calledreaction 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-therate limiting step
??How an enzyme lowers the activation energy of a reaction

Lets imagine a metal stick that should

Be broken. The energy to break it is
Represented by ∆G

The enzyme is represented by a

Magnet that binds the stick. A bad
enzyme will complement the substrate
In its initial condition. It will not be able
Break the stick. In order to do so, it will
First to break the existing magnetic contacts
And create new one. This “enzyme” will slow
.The reaction rate

The magnet weakly binds the stick. The

Magnet and stick fully complement each
Other in thetransition state when the stick
Is 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 Binding of substrates orients the substrates and allows
proximity .the active chemical groups to react

Glucose+ATP Glucose 6-phosphate

Hexokinase binds glucose and ATP

In 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
The formation of the ES complex is the faster )2
.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 ES[ is not a measurable concentration. We can[
Measure substrate and the total concentrations
Vmax=k2E t Of the enzyme. Therefore, to find the V of a
Reaction, we have to have a formula that
.use these concentrations
t ]=]E[+]ESE[[

Total Free Enzyme in

enzyme enzyme ES complex
.The reaction starts by mixing enzymes and substrates
The ES concentration builds up but quickly reaches
Asteady state. which remains almost constant
At steady state conditions
The formation of ES complex
Equals the rate of breakdown
Of the ES complex

d]ES[ =0
If all the constants are put in
One side of the equation, we can
Combine all of them to one
.Constant, Michaelis constant Km

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

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

If we insert the value of ]ES[ in the equation for velocityV=k2]ES[ We get V=k 2]]Et [ ]S

When all the enzyme is occupied in

The reaction, then V=Vmax

Therefore, we can express Michaelis

:Menten equation this way
?What could be studied from Michaelis Menten equation

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

]In this case we can neglect Km and V=Vmax]S And V=Vmax
Therefore at these conditions the velocity of the reaction is not.dependent at the concentration of the substrate

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

When Vint = )3 Vmax ]Vmax=Vmax]S

2 ]Km+]S 2
: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

Kmis 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 However, a high value of kcat will affect a high value of Km and in this case Km will
k1 .Not 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 theturnover number : the number of substrate molecules turned over per
.Enzyme molecule per second

The ratio k cat/ K Mis convenient measure of enzyme efficiency. We can understand this relation better if we consider
A 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
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/ K Mcan give
A measure for the efficiency
.And specificity of enzymes

Let us test whether a enzymatic reaction

.Behaves according to Michaelis Menten
.And to measure values for k catand KM
One can mix an enzyme and a substrate
And 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 is

Known as the Lineweaver Burk

∞=]When ]S Then 1=0 The intercept of the

]S[ graph with the coordinate
of 1/V will give the value
When we know Vmax and ]E[ t we can calculate the
Value of k cat..Because Vmax= k cat]E[ t

When V=∞ then 1/V=0. In this situation

S[=-1/K[/1M. Therefore the intercept of the graph
With the coordinate of 1/]S[ will give the value of
Enzyme Inhibition

.Many different kind of molecules inhibit enzymes by a variety of ways

A major distinction made betweenreversible 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 inhibition
:There 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 molecule
.Cannot undergo the catalytic step and therefore called an competitive inhibitor

Both substrate and inhibitor can fit in

The active site. Substrate can be processed
.By 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 would
Act 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 V.max

V max

I- I+

KM KM app ]S[

A competitive inhibitor changes the apparent KM

But does not affect V.max
Noncompetitive inhibition

When a molecule can bind to a second site on the enzyme surface in such a way that it modifies kcat. 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 k cat. The apparent
Kcat . decreases with increasing I. The change
Is actually the lower concentration of ES
Complex that is available for the catalytic
Process. Therefore V max .is affected

The apparent V maxdepends at the concentration
Of I: V max 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 of
The inhibitor affect both K Mand k cat . The latter
. Case is calledmixed 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 K Mand k cat.are affected

In this case V max,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 become
.ESI. 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

V max



KM app KM ]S[
Irreversible Inhibition

Some chemical substances can covalently

bind to the active site of enzymes and
.Block the binding of the substrate

Some of those are natural toxins and some

.Man-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 therefore
.Bind with great affinity
Sarin that is known as
Nerve gas binds to serine
In the active site of Acetyl
Cholinesterase and blocks
.Synaptic transmission

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

Penicillin inhibits
Glycopeptide transpeptidase
That forms cross links in the
Bacterial cell wall. Covalently
.Binds to Ser in active site
Enzyme activity is affected by pH

Pepsin which
Hydrolyzes peptide
.Enzymes have an optimum pH range for their activity Bonds in the stomach
Has an optimum of
.About 1.6
.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 of
.The enzyme

Glucose 6 phosphatase
The optimal pH range of an enzyme helps sometime to know what amino Functions in the liver
Acid is involved. A change in the activity around pH 7 suggest that His is Has an optimum of
.involved responsible ,7.8
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

.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 is
Achieved by the
Structure of the
Pocket. In the case
Of trypsin there
Is a negatively
Charged amino acid
.At the pocket

In Chemotrypsin
The pocket contains
Hydrophobic amino
.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 57
Is protonated. With the addition
Of substrate a proton is
Transferred from Ser 195 to
His 57 . The positively charged
Imidazole ring is stabilized by
Electrostatic interaction with
.Negatively charged Asp 102
Chemotrypsin is composed of three polypeptides linked by disulfide bonds. The active site amino acid residues
.Are 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 discriminates
.between 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 phosphorylated

Its binding induces a conformational change, and therefore the enzyme
.Is “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 Glu acts as a general

-the binding of 2 Acid catalyst, donoring
Phosphoglycerate to the A proton to the –OH
Enzyme. Residue Lys acts Leaving group The Mg ions making the C2 of phosphoglycerate which
As a general base catalyst .)Not very acidic, more acidic )lowering its pK
Receiving a proton from
: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
Allosteric interactions. The activity of many multiple subunit )1
,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

Stimulation and inhibition by control proteins: Calmodulin that )2

Senses the intracellular concentration of Ca2+ and activates many
Enzymes in the cell when Ca2+. levels rise

Reversal covalent modifications: Phosphorylation of certain amino acids in enzymes that is catalyzed by )3
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

Proteolytic activation: An irreversible conversion of an inactive to an )4

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)= other shape )enzymes that can adopt different shapes

In allosteric enzymes the binding of a substrate to one Allosteric enzymes

Active site can affect the proparties of other active sites Are represented by
.In the same emzyme molecule Sigmoidal dependence
.Cooperativity in substrate binding-Homoallostery Of reaction velocity on
.Substrate concentration
.Regulation of activity by other effector molecules-Heteroallostery

??Why this behavior is needed At low substrate

Concentration the
Enzyme has low affinity
In extreme cases enzyme can )To substrate )high Km
Regulate a substrate level to At higher substrate
Constant values. A substrate Concentration the enzyme
.That is produced all the time has high affinity )low
It accumulates to a critical level .)Km
c . The enzyme becomesS[[
Extremely efficient at this
.Substrate concentration The weak binding conformation is
This behavior helps to keep T state, and the strong binding state
.Homeostasis of dynamic systems .Is R state

Heteroallosteric effectors can be inhibitors or activators.. They bind to remote

Sites )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

The enzyme is inhibited

By the final product CTP
CTP feedback inhibition). On the)
Other hand ATP stimulates the
Enzyme. Both molecules don’t
The binding of carbamoyl Affect Vmax, but only Km. ATP
Phosphate and aspartate And CTP compete on binding to
to the enzyme is cooperative The same site on a regulatory
As can be seen by the sygmoidal .Subunit
.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 sedimentation
Coefficient of the native enzyme
Is 11.6S whereas that of the
Dissociated subunits is 2.8
And 5.8S. Therefore the two
Types of subunits can be separated
And the mercurybenzoate groups can be
.Removed by adding mercaptoethanol
The large subunits are thecatalytic ., 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
:r chains. When mixed one gets the full enzyme 2

The ATCase is composed of district catalytic and regulatory subunits

.That 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 its
Inhibitor CTP and substrates analogs. The enzyme contains a large central cavity

The regulatory subunits are found in the periphery consist

Of two domains. The outer one contains a site for CTP and the inner one
.Interacts 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 in

The center of the catalytic
Subunit. 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 transition
PALA was most important for the understanding .State
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 carbamoyl
Phosphate, 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 causes

big rotation of the catalytic and regulatory
subunits around two axis. All the catalytic
.sites are affected

)The movement of 240s loop )residues 230-245

Upon the binding of one substrate liberate
Several side chains to interact with substrates
.And 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 containing
.)Bound substrate )Y
The concerted model predicts that f R .increases more rapidly than Y

Succinate is an unreactive analog of aspartate. Y was determined

From the change in absorbance at 280 nm, and the value of f Rfrom the
.Sedimentation coefficient
The change in f Rleads the change in Y on addition of succinate, as predicted
.By 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 trimers
Were mixed with native trimers
.That can bind the substrate
Adding of succinate changed the
Absorption spectrum of nitrotyrosine
Thus, binding to one trimer changed
.The structure of the other trimer
Allosteric activator shifts the conformational
Equilibrium of all subunits to the R state, whereas
.Inhibitor shifts it towards the T state
Normal regulatory subunits were mixed with
.Nitrotyrosine containing catalytic subunits
Addition of ATP in the absence of substrate increased
The 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

Phosphatases reverse the effect of Kinases are enzymes that transfer the
Kinases by catalyzing the hydrolysis ,gamma phosphoryl group of ATP to serine
Of phosphoryl groups attached to .threonine and tyrosine residues
.The proteins

?How the phosphoryl group change the activities of proteins

It adds two negative charges to the protein. Can affect interactions with other protreins and can induce )1
.conformational changes
.The phosphoryl group can form three hydrogen bonds )2

Cyclic AMP activates protein kinase A by releasing inhibitory regulatorty subunits

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

For phosphorylation except the presence of Alanine instead of serine. This peptide
Blocks the active site from entry of substrate. cAMP releases this pseudosubstrate
.From the catralytic site
.Many enzymes are activated by specific proteolytic cleavage

Proteolytic activation of is another mechanism to activate proteins. Inactive precursors )zymogens) are activated
.By 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
Digestive enzymes in the stomach and pancreas )1

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

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

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

Developmental processes: The metamorphosis of tadpole into a frog. Large amounts of procollagen are reabsorbed )5
.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 to
Yieldα . 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 into
The anterior of the molecule and interacts with
Aspartate 194. Protonation of the amino group stabilizes
The active form the enzyme as seems by the dependence
.On enzyme activity on pH
This interaction triggers a number of conformational
.Changes 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. The
Activation occurs spontaneously below pH 5. The rate of
Cleavage is independent of the concentration of pepsinogen
.Suggesting 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 two
Chains creating an anti parallelβ . 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

Insufficientα:-antitrypsin activity cause emphysema

Antitrypsin is an plasmatic inhibitor of elastase. Elastase is a product of Neutrophils )white blood cells) . Like pancreatic
Trypsin 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 calledemphysema . 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 activation
Of the next. The many steps yield a large amplification, assuring a rapid response to

Two pathways: the intrinsic pathway is activated from non physiological surface like
.Glass 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 fibrinogen

.Into fibrin by thrombin
:Fibrinogen is made up by three globular units connected by two rods

Kd protein consists of three kinds of 340

Thrombin cleaves four arg-gly peptide bonds
In the central globular region to release an
A peptide from twoα subunits and a B peptide from twoβ . 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 by
Thrombin 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 324
Yields the active thrombin. Like in chemotrypsin, an ion pair between the positively
Charged amino group of Ile and a negatively charged group form the active site of

.Vitamin K is needed for normal clotting

Antagonists of vitamin K like Dicoumarol
And Warfarin prevent thromboses and
Used clinically. Dicoumarol treated
Animals synthesize abnormal prothrombin
That does not bind Ca2+ . The capacity to bind
.Ca resides in the amino terminus region of prothrombin
Normal prothrombin containsγ carboxyglutamate . The abnormal prothrombin formed after the administration
.)Of 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 Ca+2
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 can
.Activate fibrinogen in the plasma
.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 containing
.)VIII. 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