9/23/2011

ENZYMOLOGY

Lecture 2

Dr. S. Mukanganyama Biomolecular Interactions Analyses Lab. Department of Biochemistry University of Zimbabwe MBCHB & BDS1 2011

TEACHER: Chido, what do you call a person who keeps on talking when people are no longer interested? Tariro: A teacher/lecturer/professor etc

ENZYME KINETICS
K 1 K 2

ENZYME KINETICS
Overall rate of production of [ES] is the difference between the rates of elementary reactions leading to its appearance and those resulting in its disappearance

S
K -1

ES

P + E

(i)

d[ES]/dt =K1[E][S] - K1[ES]-K2[ES] a) Assumption of equilibrium

(iii)

General expression for the velocity (rate) of this reaction is V=d[P]/dt = K2[ES] [ ] represents concentration of the enclosed quantity. (ii)

Assuming that K1>>K2, the first step of the reaction achieves equilibrium: Ks = K-1/K1 = [E][S]/[ES] (iv)

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ENZYME KINETICS
Assumption of steady state (Briggs and Haldane,1925 ) The rate of synthesis of [ES] must equal its rate of consumption over most of the course of the reaction, i.e.[ES] is constant. Thus: d[ES]/dt = 0 (v)

ENZYME KINETICS
The quantities [ES] and [E] are not directly measurable but the total enzyme concentration ([E]T) can be determined from the following expression [E]T = [E] + [ES] (vi)

Combining (iii) with the steady state assumption (v) we get the expression: K1([E]T -[ES])[S] = (K-1 + K2)[ES] (vii)

ENZYME KINETICS
Upon rearrangement we get the following expression: [ES](K-1 + K2 + K1[S]) = K1[E]T [S] (viii) Vo

ENZYME KINETICS
The initial velocity of a reaction can be expressed in terms of experimental measurable quantities [E]T and [S] = (d[P]/dt)t = 0 = K2[ES] = K2[E]T [S]/Km + [S] (xi)

If we divide both sides by K1 and solving for [ES] [ES] = [E]T [S]/Km + [S] (ix)

The maximal velocity of a reaction Vmax occurs at high [S] when the enzyme is saturated and when it is entirely in the ES form, giving: Vmax = K2[E]T (xii)

where Km is the Michaelis-Menten constant and is defined as Km = K-1 + K2 K1 (x)

Combining (ix) and (x) we get the expression Vo = Vmax [S] Km + [S]

Michaelis-Menten equation which describes a hyperbola.

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Michaelis-Menten Graph
Approaching Zero order

First order

Plot of substrate concentration versus reaction velocity

Michaelis- Mentein kinetics

Michaelis-Menten Eq.
Allows one to easily calculate enzyme reaction velocities without having to refer to the graph every time. Two constants (specific for each enzyme) Vmax and Km, the Michaelis-Menten constant Km -substrate concentration at half the maximal velocity i.e. when [S] = Km, Vo yields Vmax/2 = Km. A measure of the affinity of the enzyme for its substrate Low Km value = High affinity, High Km value = Low Affinity

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Michaelis-Menten Equation
A small value of Km, achieves maximal catalytic efficiency at low substrate concentration. If Km and Vmax for a given enzyme are known, Michaelis-Menten equation used to calculate reaction rate, Vo. Catalytic rate constant Kcat (turnover number) of an enzyme defined as the number of molecules of substrates converted to product per molecule of enzyme per unit time

Michaelis-Menten Equation
Km -affinity of the enzyme for the substrate, Kcat -catalytic ability of the enzyme. Kcat/Km, is the specificity constant -a measure of how good the enzyme is at its job. A high specificity constant means that the reaction goes fast (Kcat is big) and does not need a high concentration of substrate (Km is small).

Lineweaver-Burk Plot
Derivation of Lineweaver-Burk plot from Michaelis-Menten equation Vo = Vmax [S] Km + [S] (a) Invert both sides of the equation we get the equation:

Lineweaver-Burk Plot

1/Vo = 1/Vo = 1/Vo = Y =

Km

[S]/Vmax[S] + 1/Vmax

(b) (c)

Km /Vmax[S]

Km /Vmax X 1/[S] + 1/Vmax (d) m x + c

Equation (d) is analogous to an equation of a straight line in the form Y = mx + c, where m = Km/Vmax, x = 1/[S], c = 1/Vmax , and Y = 1/Vo

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Lineweaver-Burk Plot
A plot of the inverse of Vo against the inverse of [S] gives Lineweaver Burk plot or Double Reciprocal plot. Gives more accurate values than the simple plot of Vo versus[S]. Michaelis-Menten equation can also be linearised to give the Eadie-Hofstee plot. Plot of Vo versus Vo/[S] gives an intercept of Vmax on the Yaxis as V/[S] tends toward zero. Slope of the line is equal to Km. The intercept on the Y-axis at Vo/[S] = Vmax/Km.

Graphical Plots
Which of these plots would one use given the values of Vo, and [S]? i) To understand the behaviour of enzymes, one uses the simple graph of initial velocity against substrate concentration. ii) Linearised forms are useful for calculation of Km and Vmax. iii) The Lineweaver-Burke plot is useful for distinguishing between the types of inhibition. iv) The Eadie-Hofstee plot is better than the LineweaverBurke plot at picking up deviations from the MichaelisMenten equation. v) Whilst, enzyme reactions are more complicated than this simple description, the Michaelis-Menten equation provides a very useful empirical tool for describing enzymes.

The Eadie-Hofstee Plot: Note that the data points are distributed much more evenly over the plot giving better statistics for the slope. In addition the value of KM is obtained from the slope, giving better precision.