ENZYME KINETICS Uni-substrate kinetics A simple model to illustrate a kinetic plot of a chemical reaction and an enzymatic reaction

Chemical reaction R v = k [R] v [R] P

Enzymatic reaction S v=? P

 Michaelis-Menten model to characterise enzyme kinetics k1 E + S k2 k1, k2, k3 = rate constants Assumptions [P] (P never accumulate Substantially) Reverse reaction does not occur 0 ES k3 E + P

v = v0 (initial rate) [E] << [S]

so that [S]0 = [S]T , [ES] is negligible

[ES] = 0 t

[ES] is constant or steady state

 Michaelis Constant Rapid equilibrium k1[E][S] = k2[ES], Michaelis-Menten when k3 << k2

[E][S] = k2 = Km [ES] k1 Steady State - Briggs-Haldane k1[E][S] = k2[ES] + k3[ES] = (k2 + k3)[ES] [E][S] = (k2 + k3) = Km [ES] k1

To derive Michaelis-Mentens Equation [E][S] = Km [ES] [E] = Km[ES] [S] - According to conservation theory of enzyme, e = [E] + [ES] = Km[ES] + [ES] [S] = [ES] ( 1 + Km ) [S] [ES] = e ( 1 + Km ) [S]

The rate of reaction ( v ) is defined as : v = [P] = k3[ES] t

- Substituting the value of [ES] : v = k3[ES] v = k3e ( 1 + Km ) [S]

Maximum rate constant ( Vm ) is attained when all the active sites are saturated with substrate, ie Vm = k3e

So that, when substituting the value of Vm v = Vm ( 1 + Km ) [S]

Rearrange the equation : v = Vm[S] [S] + Km Michaelis-Menten Equation

Validation of the Equation v =

Vm

Vm[S] [S] + Km

Vm

Km
i.

[ S ]

When [S] << Km, [S] is negligible The equation will become v = Vm[S] Km Therefore v [S] When [S] >> Km, Km is negligible v = Vm[S] = Vm [S]

ii.

iii.

when [S] = Km v = Vm[S] = Vm [S] + [S] 2

The significance of Km value

Km value is the equivalent concentration of substrate at the half of maximum rate of reaction ( Vm) If the Km value is known, then the Fraction of active site (fES) being filled up could be calculated according to the equation:
-

fES =

v Vm

[S] [S] + Km

Km is related to the rate constants of the enzymatic reactions k1 E + S k2

-

k3 E-S E + P

For steady-state Briggs-Haldane, k1[E][S] = k2[ES] + k3[ES] = (k2 + k3)[ES] [E][S] = (k2 + k3) = Km [ES] k1

For Rapid Equilibrium Michaelis-Menten k1[E][S] = k2[ES], k3 negligible because k2 >> k3 [E][S] = k2 = Km [ES] k1

In this condition :

Km = value of dissociation constant (Kdis) of ES complex The denominator of the two equations is k1 which is the value of association or formation of ES complex. Thus Km value reflects the affinity for ES complex formation or affinity/specificity of an enzyme towards substrate.
-

The lower the value of Km , the more specific/affinity an enzyme towards the substrate.

The Significance of Turnover Number (k3)

Vm e Vm = k3e k3 = Vm e = Turnover Number or Catalytic Number - It shows enzyme efficiency in catalysis of a substrate. For example : A 10-6 M Carbonic anhydrase catalyses the production of 0.6M H2CO3 in a second at substrate saturation. Thus the turnover number is 6 X 105 s-1. k3 = Vm = 0.6 = 6 X 105 s-1 e 10-6

The Importance of Km

Km can be used as an index of comparison among enzymes Km shows the specificity towards substrate Km shows the concentration of substrate / metabolites in a cell/biological system

Vm 2 V

Km [ S ]

Km could be used to determine [S] in an enzyme assay fES = v = Vm [S] [S] + Km

The normal substrate concentration is about 10 Km

-

v = 10Km = Vm 10Km + Km

10 > 90% 11

Determination Km dan Vm

Km and Vm could be determined from: a. b. c. d. Plot Michaelis-Menten Plot Lineweaver-Burk Plot Eadie-Hofstee Plot Hanes-Woolf

Plot Michaelis-Menten v = Vm[S] [S] + Km

Vm

Vm

Km

[ S ]

 Plot Lineweaver-Burk
v = Vm[S] [S] + Km

- Invert the MM equation MM 1 = [S] + Km = v Vm[S] [S] + Vm[S] Km Vm[S]

1 = Km . 1 + 1 v Vm [S] Vm
-

compare with y = mx + c

1 V

1 Vm 1

- 1 Km

[ S ]

 Plot Eadie-Hofstee
v = Vm[S] [S] + Km

1 = Km . 1 + 1 v Vm [S] Vm
-

Multiply by factor v Vm

1 (v)Vm = Km 1 v(Vm) + 1v(Vm) (v) Vm [S] Vm v = - Km . v + V m [S]

Vm

- Km V [S]

Vm Km

 Plot Hanes-Woolf
1 = Km . 1 + 1 v Vm [S] Vm - Multiply by factor [S] 1 [S] = Km 1 [S] + v Vm [S] [S] = Km + [S] v Vm Vm [S] = 1 . [S] + Km v Vm Vm 1 [S] Vm

[ S ] V Km Vm

1 Vm

[ S ] m

EISENTHAL and CORNISH-BOWDEN PLOT

Cornish-Bowden (1974) proposed a different approach to get the kinetics constants based on Michaelis-Menten equation. At a constant/fixed [E] the equation could be arranged:

1 vo Vm vo

Km

+ [So]

Vm [So] = Km + [So] [So] = Km [So]

It would rather be

+ 1

At a constant vo and [So] , the plot of Vm against Km is linear. confusing as to why a constant could be plotted against a constant.

When Km = 0 , Vm = vo and when Vm = 0 , Km = - [So]. Therefore for each pair of vo and [So] could be generated a line to note vo at Vm axis and -[So] at Km axis; the two points is joined to extrapolate a straight line. The lines for all of each pair at constant [Eo] be plotted and the intercept of the lines are the true values of Km and Vm.. However due to experimental errors the intercepts occur at a set of values (see figure). It would be logical to get the average of values or the middle of all values.

Vm Plot Eisenthal-Corning-Bowden Vm* the best estimated value Km* the best estimated value Vm* (vo)n (vo)1

Vm = Km vo + vo [ So ]

[So]n

[So]1

Km*

Km

 Reversible reaction
k1 E + S k2 ES/EP k4 k3 E + P

At equilibrium the rate of association to form the complex ES is equal to the rate dissociation of the complex ES k2 [ES] = k1 [E] [S] , that is [ES] = k1 [S] [E] k2 k3 [ES] = k4 [E] [P] , that is [ES] = k4 [P] [E] k3 From both equations, k1 [S] = k4 [P] k2 k3 [P] = k1 k3 = Keq [S] k2 k4 = Equilibrium Constant

k1 E + S k2 Vms = k3e ES

k3 E + P Kms = k2 + k3 k1 k3 EP E + P k4 Kmp = k2 + k3 k4

E + S k2 Vmp = k2e At equilibrium Vms = k3 Vmp k2

Kmp = k1 Kms k4

Keq = k1 k3 = Vms Kmp k2 k4 Vmp Kms Keq = Vms Kmp Vmp Kms Haldane Relationship

 The importance of Initial rate of reaction An important assumption to derive MM equation v = vo At the time that P is not yet accumulated. At that time the rate of reaction is proportional to the time of reaction When P could accumulate substantially, the reaction would reverse and the equations of Michaelis-Menten and Briggs-Haldane are affected k1 E + S k2 ES/EP k4 k3 E + P

the net rate of reaction for the overall reaction: vnet = k3[ES] - k2[EP]

 enzyme conservation : e = [E] + [ES] + [EP] thus, vnet = e

k3[ES] - k2[EP] [E] + [ES] + [EP]

Kms and Kmp values are : Kms = [S][E] , thus [ES] [ES] = [S][E] Kms

Kmp = [P][E] , thus[EP] = [P][E] [EP] Kmp substituting the value of [ES] and [EP] vnet = e k3[S][E] - k2[P][E] Kms Kmp [E] + [S][E] + [P][E] Kms Kmp

Vms and Vmp values : Vms = k3e Vmp = k2e

thus, vnet = Vms[S] - Vmp[P] Kms Kmp 1 + [S] + [P] Kms Kmp vnet = Vms Kmp [S] - Vmp Kms [P] Kms Kmp + Kmp [S] + Kms [P] 0 Vm[S] Km + [S]

when [P] v =

K e gradient Initial c e r u

n a

Time ( t s) a M a

Determination of the initial rate of reaction ( vo )

EISENTHAL and CORNISH-BOWDEN PLOT

Cornish-Bowden (1974) proposed a different approach to get the kinetics constants based on Michaelis-Menten equation. At a constant/fixed [E] the equation could be arranged:

1 vo Vm vo

Km

+ [So]

Vm [So] = Km + [So] [So] = Km [So]

It would rather be

+ 1

At a constant vo and [So] , the plot of Vm against Km is linear. confusing as to why a constant could be plotted against a constant.

When Km = 0 , Vm = vo and when Vm = 0 , Km = - [So]. Therefore for each pair of vo and [So] could be generated a line to note vo at Vm axis and -[So] at Km axis; the two points is joined to extrapolate a straight line. The lines for all of each pair at constant [Eo] be plotted and the intercept of the lines are the true values of Km and Vm.. However due to experimental errors the intercepts occur at a set of values (see figure). It would be logical to get the average of values or the middle of all values.

Vm Plot Eisenthal-Corning-Bowden Vm* the best estimated value Km* the best estimated value Vm* (vo)n (vo)1

Vm = Km vo + vo [ So ]

[So]n

[So]1

Km*

Km