CH13. Enzymes cXXkcZ2jWM&feature=related.

CH13. Enzymes

http://www.youtube.com/watch?v=AcXXkcZ2jWM&feature=related

Essential Questions

• What are enzymes?• What do they do?

Outline• What characteristic features define enzymes?• Can the rate of an enzyme-catalyzed reaction be defined in a

mathematical way?• What equations define the kinetics of enzyme-catalyzed

reactions?• What can be learned from the inhibition of enzyme activity?• What is the kinetic behavior of enzymes catalyzing bimolecular

reactions?• How can enzymes be so specific? • Are all enzymes proteins?• Is it possible to design an enzyme to catalyze any desired

reaction?

Virtually All Reactions in Cells Are Mediated by Enzymes

• Enzymes catalyze thermodynamically favorable reactions, causing them to proceed at extraordinarily rapid rates (see Figure 13.1)

• Enzymes provide cells with the ability to exert kinetic control over thermodynamic potentiality

• Living systems use enzymes to accelerate and control the 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 for glucose oxidation. Enzymes lower ΔG‡, thereby accelerating rate.

13.1 What Characteristic Features Define Enzymes?

• Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates

• Urease is a good example: – Catalyzed rate: 3x104/sec – Uncatalyzed rate: 3x10 -10/sec – Ratio is 1x1014

What is urease?? 5PT

Urea is the single most abundant form of dissolved organic nitrogen present in aquatic ecosystems

Specificity

• Enzymes selectively recognize proper substrates over other molecules

• Enzymes produce products in very high yields - often much greater than 95%

• Specificity is controlled by structure - the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield

Enzymes are the Agents of Metabolic Function

Figure 13.2 The breakdown of glucose by glycolysis provides a prime example of a metabolic pathway.

Enzyme Nomenclature Provides a Systematic Way of Naming Metabolic Reactions

Coenzymes and Cofactors Are Nonprotein Components Essential to Enzyme Activity

13.2 Can the Rate of an Enzyme-Catalyzed Reaction Be Defined in a Mathematical Way?

• Kinetics is the branch of science concerned with the rates of reactions

• Enzyme kinetics seeks to determine the maximum reaction velocity that enzymes can attain and binding affinities for substrates and inhibitors

• Analysis of enzyme rates yields insights into enzyme mechanisms and metabolic pathways

• This information can be exploited to control and manipulate the course of metabolic events

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 as shown:

• The rate is proportional to the concentration of A

[ ] [ ]

[ ][ ]

A P

d P d Av

dt dtA

v k Adt

Catalysts Lower the Free Energy of Activation for a Reaction

• A typical enzyme-catalyzed reaction must pass through a transition state

• The transition state sits at the apex of the energy profile in the energy diagram

• The reaction rate is proportional to the concentration of reactant molecules with the transition-state energy

• This energy barrier is known as the free energy of activation

• Decreasing ΔG‡ increases the reaction rate• The activation energy is related to the rate constant

by: /G RTk Ae

Catalysts Lower the Free Energy of Activation for a Reaction

Figure 13.5 Energy diagram for a chemical reaction (A→P) and the effects of (a) raising the temperature from T1 to T2, or (b) adding a catalyst.

The Transition State

Understand the difference between G and G‡

• The overall free energy change for a reaction is related to the equilibrium constant

• The free energy of activation for a reaction is related to the rate constant

• It is extremely important to appreciate this distinction

The Michaelis-Menten Equation is the Fundamental Equation of Enzyme Kinetics

• Louis Michaelis and Maud Menten's theory • It assumes the formation of an enzyme-substrate

complex • It assumes that the ES complex is in rapid

equilibrium with free enzyme • Breakdown of ES to form products is assumed to

be slower than 1) formation of ES and 2) breakdown of ES to re-form E and S

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

Figure 13.8 Time course for a typical enzyme-catalyzed reaction obeying the Michaelis-Menten, Briggs-Haldane models for enzyme kinetics. The early state of the time course is shown in greater magnification in the bottom graph.

[ET]=[E]+[ES]

Product formation rate=k1([ET]-[ES])[S]

[ES] dissociation=k2[ES]+k-1[ES]

d[ES]=0, steady state assumption

k1([ET]-[ES])[S] = k2[ES]+k-1[ES]

(k2+k-1)/k1 = ([ET]-[ES])[S]/[ES]

v = d[P]/dtv = k2[ES]v = k2[ET][S]/Km+[S]

Understanding Km

The "kinetic activator constant" • Km is a constant • Km is a constant derived from rate constants • Km is, under true Michaelis-Menten conditions, an

estimate of the dissociation constant of E from S • Small Km means tight binding; high Km means weak

binding

Km = (k-1+k2)/k1

Km = [S]([Et]-[ES])/[ES]

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 Turnover Number Defines the Activity of One Enzyme Molecule

A measure of catalytic activity • kcat, the turnover number, is the number of

substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate.

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

• Values of kcat range from less than 1/sec to many millions per sec

The Turnover Number Defines the Activity of One Enzyme Molecule

The Ratio kcat/Km Defines the Catalytic Efficiency of an Enzyme

The catalytic efficiency: kcat/KmAn estimate of "how perfect" the enzyme is

• kcat/Km is an apparent second-order rate constant • It measures how the enzyme performs when S is low • The upper limit for kcat/Km is the diffusion limit - the

rate at which E and S diffuse together