Lectures 5 and 6 Inhibition Systems and Derivation of their Rate Equations

1)

v=

Vm[S] S +Km =

Vmax v

v vs. [S] - Hyperbolic

2)

Lineweaver Burk - 1 v Basic Equation V = Vm[S] [S] +Km 1 v y 1 Vmax b intercept

vs

1 [S]

Linear Plot or Double Reciprocal Plot

(TAKE RECIPROCAL) Km + Vm + slope 1 [S] m X

= =

1 v

Km Vm 1 Vmax @ [S]

1 Km

1 [S]

1 [S]

O,

Km 1 Vm x [S] = O

1 Y intercept = Vmax @1 v =O

1 = Vm

Km Vm

1 S

Cross Multiply 1 1 Km = S

v EADIE-HOFSTEE PLOTS - v vs [S] V= Vm[S] [S]+Km Km 1 = Vm + Vm 1

(Gives Km & Vmax directly)

1 v

Vmax Multiply by Vmax v Slope = - Km v Vm = v + Km s Or rearranging v v = Vm - (Km) x [S] Y= b m x v [S] v Vmax Km

Rules for deriving rate laws for simple systems 1. 2. 3. Write reactions involved in forming P from S Write the conservation equation for expressing the total enzyme concentration [E]total among the various species Write the velocity dependence equation, summing all the catalytic rates constants multiplied by the concentration of the respective produt - forming species. Divide the velocity dependence equation by the conservation equation. Express the concentration of each enzymic species in terms of free enzyme concentration & substitute Algebra

4. 5. 6.

For a simple 1 step reaction, no inhibitor

(1) E+S k1[S] k2 (2) (3) (4) (5) [E]T = [E] = [ES] v = k3[ES] k3[ES] v [E]T = [E] + [ES] [ES] = k1[S] [E] - k2[ES] - k3[ES] [ES] = k1[S] [E] - (k2+ k3)[ES] @ steady state d[ES] dt = O, rate of formation = rate of breakdown ES k3 E+P

k1 [S] [E] = (k2 + k3)[ES] [ES] = k1[S][E] , k 2 + k3 Define Km = k2 + k3 k1 = [S][E] Km = [ES]

(6)

Substitute into step (4) [S] v v K3 Km [E] k3 [ET] [ET] = [S] E + Km [E] Multiply by Km v [ET] k3 [S] = ; Km + [S]

[S] v Top and bottom = k3 Km = [E ] T By[E] 1+ S Km

Define Vmax [S] Vmax = k3 [ET] ; v = Km + [S]

EQUATION FOR COMPETITIVE INNIBITION MUTUALLY EXCLUSIVE BINDING OF S AND I k1[S] k3 E +S ES E+P k2

k on [I]
EI

k off - Can drive all E to ES By increasing [S] - Since [I] & [S] are mutually exclusive binders, [I] apparently decreases affinity for E, e.g. Km

[E]T = [E] + [ES] = [EI] v = k3 [ES] v [E]T

=

k3[ES] [E]+[ES]+[EI] [S] = Km [E]

[ES] = k1 [S] [E] (k2 + k3) = [EI] =

1 Km

k on [E] [I] = k off [EI] = O; k on k off 1 KI= i.e., receprocal constant

@ steady-state d[EI] dt dissociation [EI]

[I] [E] KI [S] v = k3 Km [E] [E]T [S] E + Km [E] + [I] [E]

k3 [S] Km + [S] + Km [I] KI [I] 1 Km 1 Vmax + Vm [S] 1+ KI b+ x m

Define Vmax & Vmax [S] Double 1 Collect Terms Reciprocal = v = [I] Km 1+ KI + [S] ; Equation

DETERMINING KI FROM SLOPE REPLOT

-Measure 1 v vs 1 S @ several [I]

increasing [I] X intercept @ 1 = O v

1 Vmax 1 v [I] = O 1 v
Slope of 1 vs 1 = Km (1+ [I] s v Vm KI

= Km
Vm

1+

I KI

I S

As [I] , Slope increases

Cross multiply 1
- Km

1 Km

1+

[I] Km

1 [S]

I 1 - Km 1+ K I

1 [S]

PLOT SLOPE vs [I]

SLOPE Km Vm Km Vmax [I]

Km Km[I] SLOPE = Vmax + VmaxK I

Km Vmax KI

@ SLOPE O (i.e. x-intercept)

-Ki Km -Km = I Vmax Vmax KI Cross Multiply -KI = [I] @ slope = O

NON COMPETITIVE INHIBITION E+S k1[S] k2 k on I k off k1[S] EI + S k2 [E]T = [E] + [ES] + [EI] + [ESI] v = k3 [ES] v [E]T = k3[ES] [E] + [ES] + [EI] + [ESI] I [E] ; [EI] = KI [E] ESI ES k3 E+P

[S] [ES] = Km

[ESI] = Cannot easily calculate [ESI] by steady state hypothesis Most assume equilibrium thus is valid since EI + ESI are in equilibrium (i.e. k3 = O for ESI k2 > > > k3) THUS: [ESI] = k on [ES] I k off ; S

[I] Km

[E]

KI

v [E]T v [E]T =

S Km [E] S E+ E + [I] E + [I] Km KI KI k3 [S] Km + [S] + Km [I] + [S] KI k3 Vmax[S]

[S] E Km

v =

1+

[I] S + KI

1+

I Km KI

1 1 = Vmapp Vm

1+

I KI

Slope = Km Vm

1+

[I] KI
1 =Yint = Vmax I KI

1 v
1+ 1 Vmax //

Increasing [I] [I] =O

1 Vmaxapp

1 S

-Km I 1 1 @Xint, Vmax 1+ KI S V=O

I 1 =Vmax 1+ KI

Cross Multiply 1 1 = [S] Km 1 1 v, S Km Plot = Vm [I] KI

Replots to determine KI
Slope of 1+

Slope = y

Km + KM[I] Vm VmKI b + m x

Slope

Slope = Km VmKI
Km Vmax

x int. of slope vs [I] @ Slope = O Km[I] Km = VmK [I] I Vm

[I] -KI

Cross Multiply -KI = [I] @ Slope =O

yCan also do intercept replot as well 1 1 [I] Y int = Vmaxapp = Vmax + Vmax K I

x m

Uncompetitive Inhibition
E+S
k1[S] k2

ES

k3

E+P

I binds only to ES There is an obligate order of binding First S Then I I should decrease Km by driving reaction E+S ES towards ES formation

Like non-competitive, [S] @ cannot drive enzyme to ES form

ESI

V = k3 [ES] [E]t = [E]+[ES]+[ESI]

[ES] =

k1[S] [E] k2+k3 = 1 Km Kon[I] [ES] Koff = 1 KI k3 [ES] [E]+[ES]+[ESI]

[S] [E] Km

ESI=

I [ES] [S] [I] KI Km KI

V = [ET]

V
[ET]

S k3 Km [E] E+ S [E] + [S] [I] Km KmkI Vm [S] Km+S+ [S][I] Km+KI

Factor

Vm [S] Km+S 1+ [I] KI

Taking Reciprocal 1 V =
Km 1 1 Vm[S]+ Vm 1+[I] kI

Slope unaffected Yint decreased by [I]

Increasing [I] 1 v
1 V max 1 1 - Km [S] 1 Y int = Vmax 1 X int =Y = O; (I) 1+ KI [I] Km 1 1 Vm[S] = Vm 1+ KI I 1 1 S V = O - 1+ KI Km =

[I]=O

1 1+[I]/KI - Kmapp = Km

To obtain KI, Replot of V vs [I]

[I] 1 1 @ [S] = 1 = Vmax = Y intercept = Vmax 1+ kI v app

int = Vmax + VmaxkI - m b

1 Vmaxapp 1 KIVm 1 Vmax [I] - KI 1 @ X int = - Vmax app = O 1 [I] [] Vm = - VmaxKI

[I] - X

- KI = [I]

COMPLEX INHIBITION k1 k2 k3

E+S + I KI

ES

E+P + Ks I

Ks EI + S

k3 ESI

EI + P

= O or - Competitive = 1, = O - Non-Competitive = 1, O < < 1 - Partial Non-Competitive 1 < < , = 1 - Partial Competitive 1 < < , = O - Mixed Inhibition (Type 1) 1 < < , O < <1 - Mixed Inhibition (Type 2) In partial inhibition, the EI or ESI complexes are not dead-end complexes as they are in simply inhibition schemes

Partial Non Competitive Inhibition

k1 k3

E+S + I KI

ES
k2

E+P All forms of E (E + EI) combine equally well with S, Km does not change. Vmax is decreased because a portion of ES is ESI and ESI EI + P is slower

KI
k1

EI + S
k2

ESI

k3

E+P

V = k3 [ES] + k3 [ESI] [E]t = [E] + [ES] + [EI] + [ESI] [S] [ES] = [E] Km [EI] = [I] [E] KI [I] [S] I [ESI] = K [ES] = KI Km [E] I [S] [I] V = k3 km E + k3 K I [F]t E + () by E (x) by Km Define Vm = k3 [E]t [S] E Km

[S] [I] [S] + [I] + Km K KI I Km [S]

V=

[I] Vm 1 + KI)

_______________________

[I] [I] Km 1 + KI + [S] 1 + KI) Vm [S] [I] Km 1 + KI + [S] 1 + I 1 + KI 1+ [I] KI I KI

V=

Take Reciprocal
1 V = [I] Km 1 + KI I Vm 1 + KI 1 [S] 1 v
[I]=0 1 [S] 1 Vmax

[I] 1+ KI 1 + I Vmax 1+ KI Increasing [I] @ [I] = Ks Slope = Vm 1 Y int = Vm

1 Vmax;

[I] = l+ KI

I Ymax 1+KI

[I] 1+ KI Y intercept = I Vmax 1+ KI Simplifies to 1 1 = Y int = Ym app Vmax

[I] + KI [I] + KI 1 Limit = Vmax

Hyperbolic

Y int =

1 Vmaxapp [I]

To obtain Vmax 1 Plot Vmaxapp vs [I] Vmapp = Vmax ([I]+KI) [I] + KI

Vmapp = Vmax + VmKI [I]

multiply both terms B I+Kt I

Vmapp = Vm[I] VmKI [I]+KI [I]+KI

Partial Competitive k1 k2 k3 k2k3 Km= k1 E+P The presence of I inhibits on rate of S, Km is increased (affinity lowered) by a factor (k < 0). Since ESI, EI and E are in equilibrium, and since it makes intuitive sense that S would interfere with I binding, KI is increased by a factor 1 < < 00.

E+S + I KI

ES + I

KI Km k3

EI + S

ESI

EI

V = k3 [ES] + k3 [ESI] [E]t E + EI + ES + ESI [ES] = [S] [E] Km [EI] = [I] KI E

[I] [I] ESI = KI [ES] = KI

[S] Km

S [I] [S] [E] V = k3 Km [E] + k3 KI Km

by [E] x Km assume Vm=k3 [E]t

[S] [I] [I] [S] [E] [E]t [E] + Km + KI [E] + KI Km Factor [I] [S] KI Km [I] Vm 1 + KI S [I] Km 1 + KI [I] + S 1 + KI

[I] [S] Vm [S] + KI Km [I] Km + [S] + KI +

Vmax [S] I by 1 + KI [I] = 1 + KI Km 1 + [I] + [S] KI

0r

Vmax[S] Km App + [S]

Take Reciprocal 1 V Km = Vmax

[I] 1 + KI [I] 1 + KI

1 [S]

1 Vmax

Slope = [I] Km 1+ KI [I] Vm 1+ KI

Increasing [I]
1 V

1 Vmax -1 Kmapp

I=0

1 - Km

1 S I Km KI I Vm KI

Slope =

Km Vmax

[I] 1 + KI [I] 1 + KI

= Km + Vmax

Simplified to: Slope = Km Vm I + KI I + KI = Hyperbolic Plot

limit = k3 Vmax Slope Makes sense in that in presence of infinite [I] can still bind S, albeit weaker than in absence of I [I]