Properties of Enzymes Catalyst - speeds up attainment of reaction equilibrium Enzymatic reactions to faster than the corresponding uncatalyzed reactions Substrates - highly specific reactants for enzymes

Properties 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 Enzymes Oxidoreductases (dehydrogenases) Transferases Hydrolases Lyases Isomerases Ligases (synthetases)

1. Oxidoreductases (dehydrogenases) Catalyze oxidation-reduction reactions

2. Transferases Catalyze group transfer reactions

3. Hydrolases Catalyze hydrolysis reactions where water is the acceptor of the transferred group

4. 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. Isomerases Catalyze isomerization reactions

6. Ligases (synthetases) Catalyze ligation, or joining of two substrates Require chemical energy (e.g. ATP)

Enzyme Inhibition (Reversible) Inhibitor (I) binds to an enzyme and prevents formation of ES complex or breakdown to E + P Inhibition constant (K i ) is a dissociation constant EIE + 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 enzyme 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 enzyme inhibitors (c) Uncompetitive. I binds only to ES (inactivates E) (d) Noncompetitive. I binds to either E or ES to inactivate the enzyme

Competitive 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 for binding to trypsin

Irreversible 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 residues Reduction of a Schiff base forms a stable substituted enzyme

Inhibition of serine protease with 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 enzyme 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 Modifies 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 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 Activity

Two 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 Allosteric 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 (continued next slide)

General Properties of Allosteric 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 R 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 Regulation 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- 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 during O 2 binding to hemoglobin Oxygen binding to Hb has aspects of both the sequential and concerted models

Regulation by Covalent Modification Interconvertible enzymes are controlled by covalent modification Converter enzymes catalyze covalent modification Converter enzymes are usually controlled themselves by allosteric modulators

Pyruvate dehydrogenase regulation Phosphorylation stabilizes the inactive state (red) Dephosphorlyation stabilizes the active state (green)

Kinetic Experiments Reveal Enzyme 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 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 reaction For reactions: S 1 + S 2 P 1 + P 2 Rate is determined by the concentration of both substrates Rate equation: v = k[S 1 ] 1 [S 2 ] 1

Pseudo 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[S 1 ] 1 [S 2 ] 0 = k’[S 1 ] 1

Enzyme Kinetics Enzyme-substrate complex (ES) - complex formed when specific substrates fit into the enzyme active site E + S ESE + 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] on velocity (v) Fixed, saturating [S] Pseudo-first order enzyme-catalyzed reaction

Initial velocity (v o ) Velocity at the beginning of an enzyme-catalyzed reaction is v o (initial velocity) k 1 and k -1 represent rapid noncovalent association /dissociation of substrate from enzyme active site k 2 = rate constant for formation of product from ES E + SESE + P k1k1 k2k2 k -1

The Michaelis-Menten Equation Maximum velocity (V max ) 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 v o versus [S] curve is a rectangular hyperbola, indicating saturation of the enzyme active site as [S] increases

Plots of initial velocity (v o ) versus [S] (a) Each v o vs [S] point is from one kinetic run (b) Michaelis constant (K m ) equals the concentration of substrate needed for 1/2 maximum velocity

The Michaelis-Menten equation The Michaelis-Menten equation Equation describes v o versus [S] plots K m is the Michaelis constant V max [S] v o = K m + [S]

Derivation of the Michaelis-Menten Equation Derived from (1) Steady-state conditions: Rate of ES formation = Rate of ES decomposition (2) Michaelis constant: K m = (k -1 + k 2 ) / k 1 (3) Velocity of an enzyme-catalyzed reaction (depends upon rate of conversion of ES to E + P) v o = k 2 [ES]

The Meanings of K m K m = [S] when v o = 1/2 Vmax K m  k -1 / k 1 = K s (the enzyme-substrate dissociation constant) when k cat << either k 1 or k -1 The lower the value of K m, the tighter the substrate binding K m can be a measure of the affinity of E for S

Kinetic Constants Indicate Enzyme Activity and Specificity Catalytic constant (k cat ) - first order rate constant for conversion of ES complex to E + P k cat most easily measured when the enzyme is saturated with S Ratio k cat /K m is a second order rate constant for E + S E + P at low [S] concentrations

Meanings of k cat and k cat /K m

Measurement of K m and V max The double-reciprocal Lineweaver-Burk plot is a linear transformation of the Michaelis-Menten plot (1/v o versus 1/[S])

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

Uncompetitive inhibition

Noncompetitive inhibition