Enzyme Kinetics-2

Dr.Saba Abdi

1

Enzyme Kinetics
Enzyme Kinetics:
Study the rate of enzyme catalyzed
reactions to understand how enzyme
functions.

- Models for enzyme kinetics
- Michaelis-Menten kinetics
- Inhibition kinetics

2

Saturation
Enzyme
Kinetics
At high [S], the
enzyme is said to
be saturated with
respect to
substrate

3

Michaelis-Menten kinetics or
saturation kinetics
L. Michaelis and M.L. Menten in 1913 proposed a simple
model to account for kinetic characteristics of enzymes
• The critical feature is that a specific ES complex is a
necessary intermediate in catalysis.

• Consider an enzyme that catalyzes the S to P by the
following pathway:
k1 k2

• E + S ES E+P
k-1 k-2

4

Initial Velocity (vo )- determined from slope of
the curve at beginning of the reaction

5

6 . the reverse reaction is insignificant.Initial velocity is measured at begnning of reaction when very little P has been made. So.

M-M Enzyme Kinetics Saturation kinetics can be obtained from a simple reaction scheme that involves a reversible step for enzyme-substrate complex formation and a dissociation step of the ES complex. K1 k2 E+S K-1 ES →P + E where the rate of product formation v (moles/l-s. g/l-min) is d[P] v = = k 2 [ ES ] dt Ki is the respective reaction rate constant. 7 .

the conservation equation on the enzyme yields [ E ] = [ E0 ] − [ ES ] 8 . Enzyme Kinetics The rate of variation of ES complex is d[ES] = k1[E][S] − k−1[ES] − k2[ES] dt Since the enzyme is not consumed.

Enzyme Kinetics d [ P] v= = k [ ES ] dt 2 [ E ] = [ E0 ] − [ ES ] d[ ES ] = k1[ E ][S ] − k −1[ ES ] − k 2[ ES ] dt How to use independent variable [S] to represent v? 9 .

Briggs and Haldane Approach. .The rapid equilibrium assumption Michaelis . an assumption is required to achieve an analytical solution. 10 .The quasi-steady-state assumption. Enzyme Kinetics At this point.Menten Approach. .

K1 k2 E+S K-1 ES →P + E k1[ E ][S ] = k −1[ ES ] 11 .Assumes a rapid equilibrium between the enzyme and substrate to form an [ES] complex. Michaelis .Menten Approach The rapid equilibrium assumption: .

Menten Approach The equilibrium constant K m' can be expressed by the following equation in a dilute system. Michaelis . K1 k2 E+S ES →P + E K-1 ' k −1 [ E ][ S ] Km = = k1 [ ES ] 12 .

([ E0 ] − [ ES ])[ S ] [ ES ] == Km ' 13 .Menten Approach Then rearrange the above equation. Michaelis . [ E ][ S ] [ ES ] == Km ' Substituting [E] in the above equation with enzyme mass conservation equation [ E ] = [ E 0 ] − [ ES ] yields.

[ E0 ][ S ] [ ES ] == Km' + [S ] Then the rate of production formation v can be expressed in terms of [S].Menten Approach [ES] can be expressed in terms of [S]. d[P] k 2 [ E 0 ][ S ] Vm [ S ] v= = k 2 [ ES ] = = dt Km ' + [S ] Km' + [S ] Where Vm = k 2 [ E0 ] represents the maximum forward rate of reaction (e. Michaelis . .moles/L-min 14 ).g.

mg/L.Menten Approach K m' is often called the Michaelis-Menten constant. -The prime reminds us that it was derived by assuming rapid equilibrium in the step of enzyme-substrate complex formation. Michaelis . ' k − 1 K m = k1 15 . mol/L.

It is shown that in a closed system the quasi- steady-state hypothesis is valid after a brief transient if [S0]>> [E0].A system (batch reactor) is used in which the initial substrate concentration [S0] greatly exceeds the initial enzyme concentration [E0]. since [E0] was small. 16 . Briggs-Haldane Approach The quasi-steady-state assumption: . d[ES]/dt ≈ 0 .

The quasi-steady-state hypothesis is valid after a 17 brief transient in a batch system if [S0]>> [E0]. .

the [ES] builds up at first. when the reaction is started by mixing enzymes and substrates. This steady state will persist until almost all of the substrate has been consumed. in which [ES] remains constant. the faster ES will dissociate into E + P or E + S. • Therefore. but quickly reaches a • STEADY STATE. STEADY STATE • The more ES present. 18 .

Briggs-Haldane Approach With such assumption. the equation representing the accumulation of [ES] becomes d [ ES ] = k1[ E ][S ] − k−1[ ES ] − k2[ ES ] ≈ 0 dt Solving this algebraic equation yields k1[ E ][ S ] [ ES ] == k−1 + k2 19 .

Briggs-Haldane Approach Substituting the enzyme mass conservation equation [ E ] = [ E 0 ] − [ ES ] in the above equation yields k ([ E ] − [ ES ])[ S ] [ ES ] == 1 0 k −1 + k 2 Using [S] to represent [ES] yields [ E 0 ][ S ] [ ES ] == k −1 + k 2 + [S ] k1 20 .

2 k −1 + k 2 when K2 << k-1. Vm [ S ] v= K m + [S ] Where Vm = k [ E0 ] same as that for rapid equilibrium assumption. K m = K m ' = k −1 Km = k1 21 k 1 . Briggs-Haldane Approach Then the product formation rate becomes d [ P] k 2 [ E0 ][S ] v= = k [ ES ] = dt 2 k −1 + k 2 + [S ] k1 Then.

Km = Km '= k1 22 . Comparison of the Two Approaches Michaelis-Menten Briggs-Haldane Assumption: k1[E][S] = k−1[ES] d[ES]/dt ≈ 0 Vm [S ] Vm [ S ] Equation: v= v= Km' + [S ] K m + [S [S ] Maximum forward Vm = k 2 [ E0 ] Vm = k [ E0 ] reaction rate: 2 Constant: k −1 + k 2 ' k −1 Km = Km = k1 k1 k −1 + k 2 when k2 << k-1.

Km • Km is the [S] at 1/2 Vmax Km is a constant for a given enzyme • Km is an estimate of the equilibrium constant for S binding to E • Small Km means tight binding. high Km means weak binding 23 . high Km means • weak binding Km is the [S] at 1/2 Vmax • Km is a constant for a given enzyme • Km is an estimate of the equilibrium constant for S binding to E • Small Km means tight binding.

To reach Vmax would require that ALL enzyme molecules have tightly bound substrate . Vmax is the theoretical maximal rate of the reaction .The unit of Vm is the same as that of a reaction rate (moles/l-min.The dimension of K2 must reflect the units of [E0] • -if the enzyme is highly purified. then K2 in 1/time. Understanding Vmax- Vm =k2[E0] • The theoretical maximal velocity Vmax is a constant for a given enzyme.but it is NEVER achieved . g/l. it may be possible to express [E0] in mol/l. 24 . g/l-s) .

is the maximum number of substrate molecules converted to product per enzyme molecule per unit of time. According to M-M model. the turnover number. kcat. kcat = Vmax/Et Values of kcat range from less than 1/sec to many millions per sec 25 . The turnover number • The kcat is a direct measure of the catalytic production of product under saturating substrate conditions.

The catalytic efficiency • It shows what the enzyme can accomplish when abundant enzyme sites are available. • It is the kcat/Km value that allows direct comparison of the effectiveness of an enzyme toward different substrates. 26 .

27 .

28 .

Measuring Km and Vmax Lineweaver-Burk Plot (Double-Reciprocal Plot) Vm [ S ] v= K m + [S ] Linearizing it in double-reciprocal form: 1 1 Km 1 = + v Vm Vm S 29 .

a slope equals to Km/Vm .y-intercept is 1/Vm.More often used as it shows the independent variable [S] and dependent variable v. .Lineweaver-Burk plot (Double-reciprocal plot) .. -1/v approaches infinity as [S] decreases 30 .

y-axis intercept is Vm. -Can be subject to large error since both coordinates contain dependent variable v.the slope is –Km . . 31 but there is less bias on points at low [s]. Eadie-Hofstee Plot v v = Vm − K m [S ] .

y-axis intercept is Km/Vm .the slope is –1/Vm .better fit: even weighting of the data 32 . Hanes-Woolf (Langmuir) Plot [S ] K m 1 = + [S ] v Vm Vm .

• – Reversible: Competitive with the substrate . These can be cellular metabolites.Non-competitive with the substrate. uncompetitive and mixed inhibitor • – Irreversible (I is covalently bound. in 33 capacitating the enzyme) . or foreign substances such as drugs or toxins that have either a therapeutic or toxic (can be lethal) effect. Enzyme Inhibition • Inhibitor: Any molecule that acts directly on an enzyme to lower its catalytic rate.

to the enzyme. permanently inactivating it DIPF = DIFP = diisopropylfluorophosphate 34 . Irreversible Inhibition: inhibitor binds tightly. often covalently.

Reversible Inhibitors 35 .

i. Inhibitor COMPETES with substrate for binding. Reversible Inhibition: Competitive • Inhibitor resembles substrate but can’t undergo the catalytic step. k1 Kcat • E+S E•S E+P + k-1 I Ki EI 36 .e. so it wastes the enzyme’s time by preventing S binding.

I. Competitive inhibitor • In the presence of a competitive inhibitor. 37 . Vmax [S] v0 = Km(1 + [I]/Ki) + [S] [E][I] where Ki (inhibition constant) = [EI] Then. Vmax [S] v0 = αKm+ [S] where α = (1 + [I]/Ki) The type of inhibition can be determined using the double reciprocal plot.

Vmax does not change.app = αKm).In competitive inhibition. inhibition can be overcome by high [S]. but Km increases (Km. 38 .

Competitive inhibitor 39 .

A competitive inhibitor of dihydrofolate reductase . Is used to treat cancer 40 .Methotrexate.role in purine & pyrimidine biosynthesis.

Tetrahydrofolate-Substrate for dihydrofolate reductase 41 .

Competitive Inhibitor COO OOC H CH2 succinate dehydrogenase CH2 H COO COO Fumarate Succinate COO CH2 succinate dehydrogenase No reaction COO Malonate 42 .

Reversible Inhibition: Non- Competitive • A molecule or ion binds at a remote site on the enzyme in such a way that it affects kcat. k1 Kcat • E+S E•S E+P + k-1 + I I Ki k1 Ki EI + S ESI 43 k-1 .

44 .

noncompetitive inhibitor • Ex) Compulsory ordered Bi-Bi reaction. • B -BX E + AX X ⇄ E•AX ⇄ E•AX•B ⇄ E•A•BX ⇄ E•A ⇄ E + A B E•AXI ⇄ E•AXI•B • Compound. 45 . AXI is a noncompetitive inhibitor of B.

Uncompetitive Inhibitor • An uncompetitive inhibitor binds at a site other than the active site The inhibitor does not bind free E. • ES + I ↔ESI • Inhibitor affects both Vmax and Km. but binds E-S complex and forms an inactive E-S-I complex which cannot give normal product. 46 .

Uncompetitive Inhibitor 47 .

Uncompetitive Inhibitor • v0 = Vmax [S] • Km + α′[S] α′ where α′ = (1 + [I]/Ki′) • and Ki′ = [ES][I]/[ESI]. • Vmax.app – decrease • (by a factor of α′-1) • Km.app – decrease • (by a factor of α′-1) 48 . • uncompetitive inhibition cannot be overcome by high [S]. • • Since I does not share the binding site with S.

B ─BX E + AX X ⇄ E•AX ⇄ E•AX•B ⇄ E•A•BX ⇄ E•A ⇄ E + A E•AX•BI → No reaction Compound. Rare in single-substrate reaction. 49 . BI is an uncompetitive inhibitor of AX. More common in multisubstrate reaction • Ex) Compulsory ordered Bi-Bi reaction.

Mixes Inhibition • Inhibitor binds at a site other than the active site (E or ES) and causes changes in the overall 3-D shape of the enzyme that leads to a decrease in activity: 50 .

51 . that is.app – unchanged Vmax[S] • v0 = –––––––––– αKm + α′[S] where α = (1 + [I]/Ki) and α′ = (1 + [I]/Ki′) Ki = [E][I]/[EI]. I binds to E and ES with the same affinity (Ki = Ki′)⇒ Noncompetitive inhibition.app – decrease (by a factor of (1 + [I]/Ki)) Km. When. α = α′. Ki′ = [ES][I]/[ESI]. Mixed inhibition cannot be overcome by high [S]. Vmax.

Substrate binding Vmax is decreased unaltered. Inhibition is reversible by substrate Noncompetitive Binds E or ES complex other than at the Km appears unaltered.binding site available. Substrate binding modifies enzyme Km. Inhibitor competes with substrate for binding in a Km. decreased. Enzyme Inhibition Inhibitor Type Binding Site on Enzyme Kinetic effect Competitive Specifically at the catalytic site. making inhibitor. proportionately to but ESI complex cannot form products. Inhibition cannot be reversed by substrate 52 . is decreased structure.like process. is increased dynamic equilibrium. Inhibitor catalytic site. inhibitor concentration Inhibition cannot be reversed by substrate Uncompetitive Binds only to ES complexes at Apparent Vmax Inhibitor locations other than the catalytic site. where it Vmax is unchanged.

. 53 .

•In diagnosis of disease e. •In study of Mechanism of E action. • e.g. In prostrate cancer in men →↑Acid phosphatase • L-tartrate inhibits competitively 95% of the acid phosphate from prostrate.g.g. but has lower inhibitory effect on acid phosphatasefrom other sources. • Examples of I as drugs: •Antiviral drugs •Antibacterial drugs •Anti tumor drugs e. USES OF INHIBITORS •As drugs (in pharmacology) •Agricultural pesticides and insectidies. Samples from suspected carcinoma patients can be assessed in presence and absence of L-tartrate. sulfa drugs –Antibacterial drugs 54 .