Properties of EnzymesProperties of Enzymes

• Catalyst - speeds up attainment of reaction equilibrium

• Enzymatic reactions - 103 to 1017 faster than the corresponding uncatalyzed reactions

• Substrates - highly specific reactants for enzymes

Properties of EnzymesProperties of Enzymes

Stereospecificity - many enzymes act upon only one stereoisomer of a substrate

Reaction specificity - enzyme product yields are essentially 100% (there is no formation of wasteful byproducts)

Active site - where enzyme reactions take place

Types of EnzymesTypes of Enzymes• Oxidoreductases (dehydrogenases)

• Transferases

• Hydrolases

• Lyases

• Isomerases

• Ligases (synthetases)

1. Oxidoreductases (dehydrogenases) Catalyze oxidation-reduction reactions

2.2. TransferasesTransferases

• Catalyze group transfer reactions

3. Hydrolases3. Hydrolases

• Catalyze hydrolysis reactions where water is the acceptor of the transferred group

4. Lyases4. Lyases

Catalyze lysis of a substrate, generating a double bond in a nonhydrolytic, nonoxidative elimination (Synthases catalyze the addition to a double bond, the reverse reaction of a lyase)

5. Isomerases5. Isomerases

• Catalyze isomerization reactions

6.6. Ligases Ligases ((synthetasessynthetases))

• Catalyze ligation, or joining of two substrates

• Require chemical energy (e.g. ATP)

Enzyme Inhibition (Reversible)Enzyme Inhibition (Reversible)

• Inhibitor (I) binds to an enzyme and prevents formation of ES complex or breakdown to E + P

• Inhibition constant (Ki) is a dissociation constant EI E + I

• There are three basic types of inhibition: Competitive, Uncompetitive and Noncompetitive

• These can be distinguished experimentally by their effects on the enzyme kinetic patterns

Reversible Reversible enzyme inhibitorsenzyme inhibitors

(a) Competitive. S and I bind to same site on E

(b) Nonclassical competitive. Binding of S at active site prevents binding of I at separate site. Binding of I at separate site prevents S binding at active site.

Reversible Reversible enzyme inhibitorsenzyme inhibitors

(c) Uncompetitive. I binds only to ES (inactivates E)

(d) Noncompetitive. I binds to either E or ES to inactivate the enzyme

Competitive InhibitionCompetitive Inhibition

• Inhibitor binds only to free enzyme (E) not (ES)

• Substrate cannot bind when I is bound at active site(S and I “compete” for the enzyme active site)

• Competitive inhibitors usually resemble the substrate

Benzamidine competes with arginine Benzamidine competes with arginine for binding to trypsinfor binding to trypsin

Irreversible Enzyme InhibitionIrreversible Enzyme Inhibition

• Irreversible inhibitors form stable covalent bonds with the enzyme (e.g. alkylation or acylation of an active site side chain)

• There are many naturally-occurring and synthetic irreversible inhibitors

• These inhibitors can be used to identify the amino acid residues at enzyme active sites

• Incubation of I with enzyme results in loss of activity

Covalent complex with lysine Covalent complex with lysine residuesresidues

• Reduction of a Schiff base forms a stable substituted enzyme

Inhibition of serine protease Inhibition of serine protease with DFPwith DFP

• Diisopropyl fluorophosphate (DFP) is an organic phosphate that inactivates serine proteases

• DFP reacts with the active site serine (Ser-195) of chymotrypsin to form DFP-chymotrypsin

• Such organophosphorous inhibitors are used as insecticides or for enzyme research

• These inhibitors are toxic because they inhibit acetylcholinesterase (a serine protease that hydrolyzes the neurotransmitter acetylcholine)

Affinity labels for studying Affinity labels for studying enzyme active sitesenzyme active sites

• Affinity labels are active-site directed reagents

• They are irreversible inhibitors

• Affinity labels resemble substrates, but contain reactive groups to interact covalently with the enzyme

Site-Directed Mutagenesis Site-Directed Mutagenesis Modifies EnzymesModifies Enzymes

• Site-directed mutagenesis (SDM) can be used to test the functions of individual amino acid side chains

• One amino acid is replaced by another using molecular biology techniques

• Bacterial cells can be used to synthesize the modified protein

Practical applications of SDMPractical applications of SDM

• SDM is also used to change the properties of enzymes to make them more useful

• Subtilisin protease was made more resistant to chemical oxidation by replacing Met-222 with Ala-222 (the modified subtilisin is used in detergents)

• A bacterial protease was made more heat stable by replacing 8 of 319 amino acids

Regulation of Enzyme ActivityRegulation of Enzyme Activity

Two Methods of regulationTwo Methods of regulation

(1) Noncovalent allosteric regulation

(2) Covalent modification

• Allosteric enzymes have a second regulatory site (allosteric site) distinct from the active site

• Allosteric inhibitors or activators bind to this site and regulate enzyme activity via conformational changes

General Properties of General Properties of Allosteric EnzymesAllosteric Enzymes

1. Activities of allosteric regulator enzymes are changed by inhibitors and activators (modulators)

2. Allosteric modulators bind noncovalently to the enzymes that they regulate

3. Regulatory enzymes possess quaternary structure

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General Properties of General Properties of Allosteric EnzymesAllosteric Enzymes

4. There is a rapid transition between the active (R) and inactive (T) conformations

5. Substrates and activators may bind only to the R state while inhibitors may bind only to the T state

Rapid transition exists between Rapid transition exists between R and TR and T

• Addition of S increases concentration of the R state

• Addition of I increases concentration of the T state

• Activator molecules bind preferentially to R, leading to an increase in the R/T ratio

Two Theories of Allosteric Two Theories of Allosteric RegulationRegulation

Concerted theory - (symmetry-driven theory).

• Only 2 conformations exist: R and T ( symmetry is retained in the shift between R and T states)

• Subunits are either all R or all T

• R has high affinity for S, T has a low affinity for S

• Binding of S shifts the equilibrium toward all R state

• Binding of I shifts the equilibrium toward all T state

Sequential Theory (ligand-Sequential Theory (ligand-induced theory)induced theory)

• A ligand may induce a change in the structure of the subunit to which it binds

• Conformational change of one subunit may affect the conformation of neighboring subunits

• A mixture of both R (high S affinity) and T (low S affinity) subunits may exist (symmetry does not have to be conserved)

(a) Concerted model: subunits either all T state or all R state

(b) Sequential model: Mixture of T subunits and R subunits is possible. Binding of S converts only that subunit from T to R

Conformational changes Conformational changes during Oduring O22 binding to hemoglobin binding to hemoglobin

• Oxygen binding to Hb has aspects of both the sequential and concerted models

Regulation by Covalent Regulation by Covalent ModificationModification

• Interconvertible enzymes are controlled by covalent modification

• Converter enzymes catalyze covalent modification

• Converter enzymes are usually controlled themselves by allosteric modulators

Pyruvate dehydrogenasePyruvate dehydrogenase

regulationregulation

• Phosphorylation stabilizes the inactive state (red)

• Dephosphorlyation stabilizes the active state (green)

Kinetic Experiments Reveal Kinetic Experiments Reveal Enzyme PropertiesEnzyme Properties

Chemical Kinetics

• Experiments examine the amount of product (P) formed per unit of time ([P] / t)

• Velocity (v) - the rate of a reaction (varies with reactant concentration)

• Rate constant (k) - indicates the speed or efficiency of a reaction

First order rate equationFirst order rate equation

• Rate for nonenzymatic conversion of substrate (S) to product (P) in a first order reaction: (k is expressed in reciprocal time units (s-1))

[P] / t = v = k[S]

Second order reactionSecond order reaction

• For reactions: S1 + S2 P1 + P2

• Rate is determined by the concentration of both substrates

• Rate equation: v = k[S1]1[S2]1

Pseudo first order reactionPseudo first order reaction

• If the concentration of one reactant is so high that it remains essentially constant, reaction becomes zero order with respect to that reactant

• Overall reaction is then pseudo first-order

v = k[S1]1[S2]0 = k’[S1]1

Enzyme KineticsEnzyme Kinetics• Enzyme-substrate complex (ES) - complex

formed when specific substrates fit into the enzyme active site

E + S ES E + P

• When [S] >> [E], every enzyme binds a molecule of substrate (enzyme is saturated with substrate)

• Under these conditions the rate depends only upon [E], and the reaction is pseudo-first order

Effect of enzyme concentration [E] Effect of enzyme concentration [E] on velocity (v)on velocity (v)

• Fixed, saturating [S]

• Pseudo-first order enzyme-catalyzed reaction

Initial velocity (vInitial velocity (voo))

• Velocity at the beginning of an enzyme-catalyzed reaction is vo (initial velocity)

• k1 and k-1 represent rapid noncovalent association /dissociation of substrate from enzyme active site

• k2 = rate constant for formation of product from ES E + S ES E + P

k1 k2

k-1

The Michaelis-Menten EquationThe Michaelis-Menten Equation

• Maximum velocity (Vmax) is reached when an enzyme is saturated with substrate (high [S])

• At high [S] the reaction rate is independent of [S] (zero order with respect to S)

• At low [S] reaction is first order with respect to S

• The shape of a vo versus [S] curve is a rectangular hyperbola, indicating saturation of the enzyme active site as [S] increases

Plots of initial Plots of initial velocity (vvelocity (voo) versus [S]) versus [S]

(a) Each vo vs [S] point is from one kinetic run

(b) Michaelis constant (Km) equals the concentration of substrate needed for 1/2 maximum velocity

The Michaelis-Menten The Michaelis-Menten equationequation

• Equation describes vo versus [S] plots

• Km is the Michaelis constant

Vmax[S]

vo =

Km + [S]

Derivation of the Derivation of the Michaelis-Menten EquationMichaelis-Menten Equation

• Derived from

(1) Steady-state conditions:

Rate of ES formation = Rate of ES decomposition

(2) Michaelis constant: Km = (k-1 + k2) / k1

(3) Velocity of an enzyme-catalyzed reaction (depends upon rate of conversion of ES to E + P)

vo = k2[ES]

The Meanings of KThe Meanings of Kmm

• Km = [S] when vo = 1/2 Vmax

• Km k-1 / k1 = Ks (the enzyme-substrate dissociation constant) when kcat << either k1 or k-1

• The lower the value of Km, the tighter the substrate binding

• Km can be a measure of the affinity of E for S

Kinetic Constants Indicate EnzymeKinetic Constants Indicate Enzyme Activity and Specificity Activity and Specificity

• Catalytic constant (kcat) - first order rate constant for conversion of ES complex to E + P

• kcat most easily measured when the enzyme is saturated with S

• Ratio kcat /Km is a second order rate constant for E + S E + P at low [S] concentrations

Meanings of kMeanings of kcatcat and k and kcatcat/K/Kmm

Measurement of KMeasurement of Kmm and V and Vmaxmax

The double-reciprocal Lineweaver-Burk plot is a linear transformation of the Michaelis-Menten plot

(1/vo versus 1/[S])

Competitive inhibition: Competitive inhibition: (a) Kinetic scheme. (a) Kinetic scheme. (b) Lineweaver-Burk plot(b) Lineweaver-Burk plot

Uncompetitive inhibitionUncompetitive inhibition

Noncompetitive inhibitionNoncompetitive inhibition