chapter 6 mckee enzyme kinetics

04/08/23Prentice Hall c2002

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Properties 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


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Properties of enzymes (continued)

• 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


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The Six Classes of Enzymes

1. Oxidoreductases (dehydrogenases)

• Catalyze oxidation-reduction reactions


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2. Transferases

• Catalyze group transfer reactions


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3. Hydrolases

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


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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)


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5. Isomerases

• Catalyze isomerization reactions


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6. Ligases (synthetases)

• Catalyze ligation, or joining of two substrates

• Require chemical energy (e.g. ATP)


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Kinetic Experiments Reveal Enzyme Properties

A. 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


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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]


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Second 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


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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[S1]1[S2]0 = k’[S1]1


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B. Enzyme Kinetics

• 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

• Enzyme-substrate complex (ES) - complex formed when specific substrates fit into the enzyme active site

E + S ES E + P


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

• Fixed, saturating [S]

• Pseudo-first order enzyme-catalyzed reaction


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Initial velocity (vo)

• 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


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Progress curve for an enzyme-catalyzed reaction

• The initial velocity (vo) is the slope of the initial linear portion of the curve

• Rate of the reaction doubles when twice as much enzyme is used


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The 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


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Plots of initial velocity (vo) 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


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The Michaelis-Menten equation

• Equation describes vo versus [S] plots

• Km is the Michaelis constant

Vmax[S]

vo =

Km + [S]


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A. Derivation of the Michaelis-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]


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B. The Meanings of Km

• 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


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Kinetic Constants Indicate Enzyme 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 (region A on Figure 5)

• Ratio kcat /Km is a second order rate constant for E + S E + P at low [S] concentrations (region B on Figure 5)


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Meanings of kcat and kcat/Km

Catalytic Efficiency

Rate of Conversion


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Values of kcat/Km

• kcat/Km can approach rate of encounter of two uncharged molecules in solution (108 to 109M-1s-1)

• kcat/Km is also a measure of enzyme specificity for different substrates (specificity constant)

• rate acceleration = kcat/kn

(kn = rate constant in the absence of enzyme)


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Measurement of Km and Vmax

Fig 5.6 The double-reciprocal Lineweaver-Burk plot is a linear transformation of the Michaelis-Menten plot

(1/vo versus 1/[S])


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Reversible Enzyme Inhibition

• 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


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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.


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Reversible enzyme inhibitors(cont)

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

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


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A. 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)

• Vmax is the same with or without I (high S can still saturate the enzyme even in the presence of I)

• Apparent Km (Kmapp) measured in the presence of I is

larger than KM (measured in absence of I)

• Competitive inhibitors usually resemble the substrate


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


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B. Uncompetitive Inhibition

• Uncompetitive inhibitors bind to ES not to free E

• Vmax decreased by conversion of some E to ESI

• Km (Kmapp) is also decreased

• Lines on double-reciprocal plots are parallel

• This type of inhibition usually only occurs in multisubstrate reactions


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Uncompetitive inhibition


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C. Noncompetitive Inhibition

• Noncompetitive inhibitors bind to both E and ES

• Inhibitors do not bind at the same site as S

• Vmax decreases

• Km does not change

• Inhibition cannot be overcome by addition of S

• Lines on double-reciprocal plot intersect on x axis


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Noncompetitive inhibition


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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


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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)


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• Reaction of DFP with Ser-195 of chymotrypsin


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Coenzymes and Vitamins

Apoenzyme + Cofactor Holoenzyme

(protein only) (active)

(inactive)

• Some enzymes require cofactors for activity

(1) Essential ions (mostly metal ions)

(2) Coenzymes (organic compounds)


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Coenzymes

• Coenzymes act as group-transfer reagents

• Hydrogen, electrons, or other groups can be transferred

• Larger mobile metabolic groups can be attached at the reactive center of the coenzyme

• Coenzyme reactions can be organized by their types of substrates and mechanisms


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Types of cofactors


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Many Enzymes Require Inorganic Cations

• Enzymes requiring metal ions for full activity:

(1) Metal-activated enzymes have an absolute requirement or are stimulated by metal ions (examples: K+, Ca2+, Mg2+)

(2) Metalloenzymes contain firmly bound metal ions at the enzyme active sites (examples:

iron, zinc, copper, cobalt )


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Protein Coenzymes

• Protein coenzymes (group-transfer proteins) contain a functional group as part of a protein or as a prosthetic group

• Participate in:(1) Group-transfer reactions (2) Oxidation-reduction reactions where transferred group is a hydrogen or an electron

• Metal ions, iron-sulfur clusters and heme groups are commonly found in these proteins


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Coenzyme Classification

• There are two classes of coenzymes

(1) Cosubstrates are altered during the reaction and regenerated by another enzyme

(2) Prosthetic groups remain bound to the enzyme during the reaction, and may be

covalently or tightly bound to enzyme


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Classification of coenzymes in mammals

(1) Metabolite coenzymes - synthesized from common metabolites

(2) Vitamin-derived coenzymes - derivatives of vitamins (vitamins cannot be synthesized by mammals, but must be obtained as nutrients)


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A. Metabolite Coenzymes

• Nucleoside triphosphates are examples

ATP


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Reactions of ATP

• ATP is a versatile reactant that can donate its:

(1) Phosphoryl group (-phosphate)

(2) Pyrophosphoryl group ( phosphates)

(3) Adenylyl group (AMP)

(4) Adenosyl group


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B. Vitamin-Derived Coenzymes and Nutrition

• Vitamins are required for coenzyme synthesis and must be obtained from nutrients

• Animals rely on plants and microorganisms for vitamin sources (meat supplies vitamins also)

• Most vitamins must be enzymatically transformed to the coenzyme


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Oxidized, reduced forms of NAD (NADP)


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NAD and NADP are cosubstrates for dehydrogenases

• Oxidation by pyridine nucleotides always occurs two electrons at a time

• Dehydrogenases transfer a hydride ion (H:-) from a substrate to pyridine ring C-4 of NAD+ or NADP+

• The net reaction is:

NAD(P)+ + 2e- + 2H+ NAD(P)H + H+


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FAD and FMN

• Flavin adenine dinucleotide (FAD) and Flavin mono-nucleotide (FMN) are derived from riboflavin (Vit B2)

• Flavin coenzymes are involved in oxidation-reduction reactions for many enzymes (flavoenzymes or flavoproteins)

• FAD and FMN catalyze one or two electron transfers


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Fig 7.12 Reduction, reoxidation of FMN or FAD


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Coenzyme A (CoA or HS-CoA)

• Derived from the vitamin pantothenate (Vit B3)

• Participates in acyl-group transfer reactions with carboxylic acids and fatty acids

• CoA-dependent reactions include oxidation of fuel molecules and biosynthesis of carboxylic acids and fatty acids

• Acyl groups are covalently attached to the -SH of CoA to form thioesters


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Coenzyme A


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Ubiquinone (Coenzyme Q)

• Found in respiring organisms and photosynthetic bacteria

• Transports electrons between membrane-embedded complexes

• Plastoquinone (ubiquinone analog) functions in photosynthetic electron transport


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(a) Ubiquinone, (b) Plastoquinone

• Hydrophobic tail of each is composed of 6 to 10 five-carbon isoprenoid units

• The isoprenoid chain allows these quinones to dissolve in lipid membranes


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Cytochromes

• Heme-containing coenzymes whose Fe(III) undergoes reversible one-electron reduction

• Cytochromes a,b and c have different visible absorption spectra and heme prosthetic groups

• Electron transfer potential varies among different cytochromes due to the different protein environment of each prosthetic group


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(a) Heme group of cyt a


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(b) Heme group of cyt b


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(c) Heme group of cyt c


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Temperature vs. Enzyme Activity

Regulation


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pH vs. Enzyme Activity


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Regulation of Enzyme Activity

• Regulatory enzymes - activity can be reversibly modulated by effectors

• Such enzymes are usually found at the first unique step in a metabolic pathway (the first “committed” step)

• Regulation at this step conserves material and energy and prevents accumulation of intermediates


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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


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A. 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


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General scheme for covalent modification of interconvertible enzymes


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Pyruvate dehydrogenase regulation

• Phosphorylation stabilizes the inactive state (red)

• Dephosphorlyation stabilizes the active state (green)


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B. Phosphofructokinase-1 (PFK-1) Is an Allosteric Enzyme

• PFK-1catalyzes an early step in glycolysis (an ATP-generating pathway for glucose degradation)

• Phosphoenol pyruvate (PEP), an intermediate near the end of the pathway is an allosteric inhibitor of PFK-1

• ADP is an allosteric activator of PFK-1


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Regulation of glycolysis by PFK-1

• When the ratio [PEP]/[ADP] is high, flux through glycolysis decreases at the PFK-1 step

• When the ratio of [PEP]/[ADP] is low, PFK-1 is activated and glycolysis produces more ATP from ADP

• Thus the concentrations of PEP and ADP act allosterically through PFK-1 to regulate the activity of the entire pathway


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Reaction catalyzed by PFK-1


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Phosphoenolpyruvate (PEP)


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Effect of PEP and ADP on PFK-1

• Both PEP and ADP affect the binding of the substrate fructose 6-phosphate (F6P) to PFK-1

• PFK-1 exhibits a sigmoidal (S-shaped) vo versus [S] curve. This is due to the cooperativity of S binding.

• Increasing ADP concentrations lowers the apparent Km for F6P without affecting Vmax (activates PFK)

• The allosteric inhibitor PEP raises the apparent Km without changing Vmax


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Plots of initial velocity versus F6P for PFK-1

• ADP is an allosteric activator of PFK-1 and lowers the apparent Km without affecting Vmax

• For a given F6P concentration the vo is larger in the presence of ADP


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C. 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)


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General properties of regulatory enzymes (cont)

4. Regulatory enzymes have at least one substrate which has a sigmoidal vo versus [S] curve (positive cooperativity of multiple substrate binding sites)

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

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


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Rapid transition exists between R and T states

• 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


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Role of cooperativity of binding in regulation

• Addition of modulators alters enzyme activity

• Activators can lower Km, inhibitors can raise Km


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D. 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


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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)


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Two models

(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

chapter 6 mckee enzyme kinetics

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