Chem 4311- Chapter13-15 Enzyme Cropped

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Chapter 13-15Enzyme

EXAM - October 19

Instructor: Dr. Khairul I AnsariOffice: 316CPBPhone: 817-272-0616email: [email protected] hours 12 am 1:30 pm Tuesday &.Thursday

CHEM 4311

General Biochemistry I

Fall 2012

Chapter 13

Enzyme Kinetics


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Virtually All Reactions in Cells Are Mediated by

Enzymes

Enzymes catalyze thermodynamically favorablereactions, causing them to proceed at extraordinarilyrapid rates

Enzymes provide cells with the ability to exert kineticcontrol over thermodynamic potentiality

Living systems use enzymes to accelerate and controlthe rates of vitally important biochemical reactions

Enzymes are the agents of metabolic function

Virtually All Reactions in Cells Are Mediated by

Enzymes

Figure 13.1 Reaction profile showing the large free energy of activation forglucose oxidation. Enzymes lower G, thereby accelerating rate.

C6H12O6 + 6O2 6CO2 + 6H20 + 2870kJ Energy


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13.1 What Characteristic Features Define Enzymes?

Catalytic power is defined as the ratio of the enzyme-catalyzedrate of a reaction to the uncatalyzed rate

Specificity is the term used to define the selectivity of enzymes fortheir substrates

Regulation of enzyme activity ensures that the rate of metabolicreactions is appropriate to cellular requirements

Enzyme nomenclature provides a systematic way of namingmetabolic reactions

Coenzymes and cofactors are nonprotein components essential toenzyme activity.

13.1 What Characteristic Features Define Enzymes?

Enzymes can accelerate reactions as much as 1021

over uncatalyzed rates

Urease is a good example:

Catalyzed rate: 3x104

/secUncatalyzed rate: 3x10 -10/sec

Ratio (catalytic power) is 1x1014


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Several Kinetics Terms to Understand

rate or velocity

rate constant

rate law

order of a reaction

molecularity of a reaction

Chemical Kinetics Provides a Foundation for Exploring

Enzyme Kinetics

Consider a reaction of overall stoichiometry asshown:

The rate is proportional to the concentrationof A

A P

v =d[P]

dt

=d[A]

dt

v =[A]

dt= k[A]


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Catalysts Lower the Free Energy of Activation for a Reaction

A typical enzyme-catalyzed reaction must pass through atransition state

The transition state sits at the apex of the energy profilein the energy diagram

The reaction rate is proportional to the concentration ofreactant molecules with the transition-state energy

This energy barrier is known as the free energy ofactivation

Decreasing G increases the reaction rate

The activation energy is related to the rate constant by:

k= eG/RT

Catalysts Lower the Free Energy of Activation for a Reaction

Figure 13.5 Energy diagram for a chemical reaction (AP)and the effects of (a) raising the temperature from T1 to T2, or

(b) adding a catalyst.


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13.3 What Equations Define the Kinetics of Enzyme-

Catalyzed Reactions?

Figure 13.6 A plot of versus [A] for the unimolecular chemical reaction,AP, yields a straight line having a slope equal to k. This reaction is a first-order reaction.

As [S] increases, kinetic behavior changes from 1st order to

zero-order kinetics

Figure 13.7 Substrate saturationcurve for an enzyme-catalyzed

reaction.


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The Michaelis-Menten Equation is the Fundamental

Equation of Enzyme Kinetics

Louis Michaelis and Maud Menten's theory

assumes the formation of an enzyme-substratecomplex assumes that the ES complex is in rapid equilibrium

with free enzyme assumes that the breakdown of ES to form products

is slower than

1) formation of ES and2) breakdown of ES to re-form E and S

[ES] Remains Constant Through Much of the Enzyme ReactionTime Course in Michaelis-Menten Kinetics

Figure 13.8 Time course for atypical enzyme-catalyzed reactionobeying the Michaelis-Menten,Briggs-Haldane models forenzyme kinetics. The early state

of the time course is shown ingreater magnification in thebottom graph.


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Understanding Vmax

The theoretical maximal velocity

Vmax is a constant Vmax is the theoretical maximal rate of the reaction -

but it is NEVER achieved in reality To reach Vmax would require that ALL enzyme

molecules are tightly bound with substrate Vmax is asymptotically approached as substrate is

increased

The dual nature of the Michaelis-Menten equation

Combination of 0-order and 1st-order kinetics

When S is low, the equation for rate is 1st order in S

When S is high, the equation for rate is 0-order in S

The Michaelis-Menten equation describes a rectangularhyperbolic dependence of v on S


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The Turnover Number Defines the Activity of OneEnzyme Molecule

A measure of catalytic activity

kcat, the turnover number, is the number of substratemolecules converted to product per enzymemolecule per unit of time, when E is saturated withsubstrate.

If the M-M model fits, k2 = kcat = Vmax/Et

Values ofkcat range from less than 1/sec to manymillions per sec

The Turnover Number Defines the Activity of One Enzyme

Molecule

Turn over number (Kca.s-1) of

Catalase 40,000,000DNA Plymerase I 15


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In an Ordered, Single-Displacement Reaction, the Leading

Substrate Must Bind First

The leading substrate (A) binds first, followed by B.

Reaction between A and B occurs in the ternary complexand is usually followed by an ordered release of theproducts, P and Q.

An Alternative way of Portraying the Ordered, Single-

Displacement Reaction


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Glutamate:aspartate Aminotransferase

Figure 13.23An enzymeconforming to adouble-

displacementbisubstratemechanism.

13.7 How Can Enzymes Be So Specific?

The

Lock and key

hypothesis was the first explanationfor specificity

The Induced fit hypothesis provides a more accuratedescription of specificity

Induced fit favors formation of the transition state

Specificity and reactivity are often linked. In thehexokinase reaction, binding of glucose in the active siteinduces a conformational change in the enzyme thatcauses the two domains of hexokinase to close aroundthe substrate, creating the catalytic site


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13.7 How Can Enzymes Be So Specific?

Figure 13.24 A drawing, roughly to scale, of H2O, glycerol,glucose, and an idealized hexokinase molecule. Binding ofglucose in the active site induces a conformational changein the enzyme that causes the two domains of hexokinaseto close around the substrate, creating the catalytic site.

13.7 Are All Enzymes Proteins?

Ribozymes - segments of RNA that display enzyme

activity in the absence of proteinExamples: RNase P and peptidyl transferase

Abzymes - antibodies raised to bind the transition stateof a reaction of interest


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Chapter 14Mechanisms of Enzyme Action

14.1 What Are the Magnitudes of Enzyme-InducedRate Accelerations?

Enzymes are powerful catalysts

The large rate accelerations of enzymes (107 to 1015)correspond to large changes in the free energy ofactivation for the reaction

All reactions pass through a transition state on the

reaction pathway

The active sites of enzymes bind the transition stateof the reaction more tightly than the substrate

By doing so, enzymes stabilize the transition stateand lower the activation energy of the reaction


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14.2 What Role Does Transition-State Stabilization Play in

Enzyme Catalysis?

The catalytic role of an enzyme is to reduce the energy barrier betweensubstrate S and transition state X

Rate acceleration by an enzyme means that the energy barrier between

ES and EX

must be smaller than the barrier between S and X

This means that the enzyme must stabilize the EX transition state morethan it stabilizes ES

14.3 How Does Destabilization of ES Affect Enzyme

Catalysis?

Raising the energy of ES raises the rate

For a given energy of EX, raising the energy of ES willincrease the catalyzed rate

This is accomplished by

a) loss of entropy due to formation of ESb) destabilization of ES by

strain

distortion

desolvation


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Catalysis?

Figure 14.2 The intrinsic binding energy of ES is compensated by entropyloss due to binding of E and S and by destabilization due to strain anddistortion.


Catalysis?

Figure 14.3 (a) Catalysis does not occur if ES and X

are equallystabilized. (b) Catalysis willoccur if X is stabilized more than ES.


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14.3 How Does Destabilization of ES AffectEnzyme Catalysis?

Figure 14.4 (a) Formation of the ES complex results in entropyloss. The ES complex is a more highly ordered, low-entropystate for the substrate.

14.3 How Does Destabilization of ES Affect EnzymeCatalysis?

Figure 14.4 (b) Substrates typically lose waters of hydrationin the formation in the formation of the ES complex.

Desolvation raises the energy of the ES complex, making itmore reactive.


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Transition-State Analogs Make Our World Better

Enzymes are often targets for drugs and other beneficialagents

Transition-state analogs often make ideal enzyme inhibitors Enalapril and Aliskiren lower blood pressure

Statins lower serum cholesterol

Protease inhibitors are AIDS drugs

Juvenile hormone esterase is a pesticide target

Tamiflu is a viral neuraminidase inhibitor


Statins such as Lipitor arepowerful cholesterol-loweringdrugs, because they aretransition-state analog inhibitors

of HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A )

reductase, a key enzyme in thebiosynthetic pathway forcholesterol.


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One strategy for controlling insect populations is to alter theactions of juvenile hormone, a terpene-based substance thatregulates insect life cycle processes. Levels of juvenilehormone are controlled by juvenile hormone esterase (JHE),and inhibition of JHE is toxic to insects. OTEP (figure) is apotent transition state-analog inhibitor of JHE.

Tamiflu is a Viral Neuraminidase Inhibitor

Influenza is a serious illness that

affects 5% to 15% of the earthspopulation each year and results inup to 500,000 deaths annually. Neuraminidase is a majorglycoprotein on the influenza virusmembrane envelope that isessential for viral replication andinfectivity. Tamiflu is a neuraminidaseinhibitor and antiviral agent basedon the transition state of the

neuraminidase reaction.


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Protein Motions Are Essential to Enzyme Catalysis

Figure 14.10 Catalysis in enyzme active sites depends on motionof active-site residues. Several active-site residues undergogreater motion during catalysis than residues elsewhere in the

protein.

Mechanisms of Catalysis

1. Covalent Catalysis

Some enzymes derive much of their rate acceleration fromformation of covalent bonds between enzyme and substrate

The side chains of amino acids in proteins offer a variety ofnucleophilic centers for catalysis

These groups readily attack electrophilic centers ofsubstrates, forming covalent enzyme-substrate complexes

The covalent intermediate can be attacked in a second step bywater or by a second substrate, forming the desired product


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4. Quantum Mechanical Tunneling

Tunneling provides a path around the usual energyof activation for steps in chemical reactions

Many enzymes exploit this

According to quantum theory, there is a finiteprobability that any particle can appear on the otherside of an activation barrier for a reaction step

The likelihood of tunneling depends on the distance

over which a particle must move

Only protons and electrons have a significantprobability of tunneling

Tunneling between donor and acceptor

Figure 14.13d If the distance for particle transfer is sufficientlysmall, overlap of probability functions (red) permit efficientquantum mechanical tunneling between donor (D) and acceptor

(A)


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5. Metal Ion Catalysis

Figure 14.14 Thermolysin is an endoprotease with a catalytic Zn2+ ion inthe active site. The Zn2+ ion stabilizes the buildup of negative charge onthe peptide carbonyl oxygen, as a glutamate residue deprotonates water,promoting hydroxide attack on the carbonyl carbon.

Reaction by MetalloenzymeEg Thermolysin and Zn

How Do Active-Site Residues Interact to Support

Catalysis?

About half of the amino acids engage directly in catalyticeffects in enzyme active sites

Other residues may function in secondary roles in the activesite:

Raising or lowering catalytic residue pKa

values

Orientation of catalytic residues

Charge stabilization

Proton transfers via hydrogen tunneling


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Aspartic proteases show one relatively low pKa, and onerelatively high pKa

This was once thought to represent pKa values of the twoaspartate residues, but this is no longer believed to be the case

Instead, molecular dynamics simulations show that asparticproteases employ low-barrier hydrogen bonds (LBHBs) in theirmechanism

The predominant catalytic factor in aspartic proteases is generalacid-base catalysis

Protease inhibitors as AIDS drugs

If HIV-1 protease can be selectively inhibited, then new HIVparticles cannot form

Several novel protease inhibitors are currently marketed as AIDSdrugs

Many such inhibitors work in a culture dish However, a successful drug must be able to kill the virus in a

human subject without blocking other essential proteases in thebody


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Two prominent way to regulate enzyme

1. By increasing or decreasing enzyme molecules-byregulating gene expression (induction or suppression)and protein degradation

2. Increasing or decreasing intrinsic activity of each enzymemolecules-principally by allosteric regulation or covalent

modifications

Allosteric Regulation? In allosteric regulation (feedback control) the effector molecule

binds at the protein's allosteric site (that is, a site other than the

protein's active site). If the effectors that enhance the activity - allosteric activators If the effectors that decrease the activity -allosteric inhibitors. Enzymes situated at key steps in metabolic pathways are modulated

by allosteric effectors These effectors are usually produced elsewhere in the pathway

Features of allosteric regulation1. Does not follow Michaelis-Menten equation2. Feedback inhibition is different than normal enzyme inhibitor3. The effector molecules may activate the allosteric enzyme or

stimulate its activity4. Most allosteric enzymes have oligomeraic organization

5. The interaction of the enzyme with effector molecule causesconformation changes in the active site of the enzyme.


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Enzyme activity can be regulated by covalent Modification

eg Phosphorylation, acetylation etc

The interconvertible enzymes can by reversibly modified

Enzyme activity can be regulated through reversible phosphorylation This is the most prominent form of covalent modification in cellular

regulation

Phosphorylation is accomplished by protein kinases

Each protein kinase targets specific proteins for phosphorylation

Phosphoprotein phosphatases catalyze the reverse reaction

removing phosphoryl groups from proteins Kinases and phosphatases themselves are targets of regulation

15.4 What Kinds of Covalent Modification Regulate the

Activity of Enzymes?

Protein kinases phosphorylate Ser, Thr, and Tyr residues in targetproteins

Kinases typically recognize specific amino acid sequences in theirtargets

In spite of this specificity, all kinases share a common catalyticmechanism based on a conserved core kinase domain of about

260 residues (see Figure 15.9) Kinases are often regulated by intrasteric control, in which a

regulatory subunit (or domain) has a pseudosubstrate sequencethat mimics the target sequence, minus the phosphorylatableresidue


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Isozymes Are Enzymes With Slightly Different

Subunits

Figure 15.5 Theisozymes of lactatedehydrogenase (LDH).

15.5 Are Some Enzymes Controlled by Both Allosteric

Regulation and Covalent Modification?

Glycogen phosphorylase (GP) is an example of the manyenzymes that are regulated both by allosteric controls andby covalent modification

GP cleaves glucose units from nonreducing ends ofglycogen

This converts glycogen into readily usable fuel in the formof glucose-1-phosphate

This is a phosphorolysis reaction

Muscle GP is a dimer of identical subunits, each with PLPcovalently linked

There is an allosteric effector site at the subunit interface


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Glycogen Phosphorylase Activity is Regulated

Allosterically

Muscle glycogen phosphorylase shows cooperativity insubstrate binding

ATP and glucose-6-P are allosteric inhibitors of glycogenphosphorylase

AMP is an allosteric activator of glycogen phosphorylase

When ATP and glucose-6-P are abundant, glycogenbreakdown is inhibited

When cellular energy reserves are low (i.e., high [AMP]and low [ATP] and [G-6-P]) glycogen catabolism isstimulated

Glycogen Phosphorylase Activity is RegulatedAllosterically

Figure 15.15 v versus S curves for glycogen phosphorylase.(a)The response to the concentration of the substratephosphate (Pi).(b)ATP is a feedback inhibitor.(c)AMP is a positive effector. It binds at the same site as ATP.


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Fe2+ is coordinated by His F8

Iron interacts with six ligands in Hb and Mb

Four of these are the N atoms of the porphyrin A fifth ligand is donated by the imidazole side chain of amino

acid residue His F8

(This residue is on the sixth or F helix, and it is the 8th residuein the helix, thus the name.)

When Mb or Hb bind oxygen, the O2 molecule adds to the heme

iron as the sixth ligand The O2 molecule is tilted relative to a perpendicular to the heme

plane

Myoglobin Structure

Mb is a monomeric heme protein Mb polypeptide "cradles" the heme group Fe in Mb is Fe2+ - ferrous iron - the form that binds

oxygen Oxidation of Fe yields 3+ charge - ferric iron Mb with Fe3+ is called metmyoglobin and does not

bind oxygen


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Fe2+ Movement by Less Than 0.04 nm Induces the

Conformation Change in Hb

In deoxy-Hb, the iron atom lies out of the heme plane by about0.06 nm

Upon O2 binding, the Fe2+ atom moves about 0.039 nm closer to the

plane of the heme

It is as if the O2 is drawing the heme iron into the plane

This may seem like a trivial change, but its biological consequencesare far-reaching

As Fe2+

moves, it drags His F8 and the F helix with it This change is transmitted to the subunit interfaces, where

conformation changes lead to the rupture of salt bridges

Fe2+ Movement by Less Than 0.04 nm Induces theConformation Change in Hb


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Chem 4311- Chapter13-15 Enzyme Cropped

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