LECTURES812:EnzymeKinetics

Problem 1
Determine the initial rate of product (P) formation from enzyme X and
substrate Y that form the complex XY. The reaction kinetics is given by

Solution:
Michaelis-Menten approach : The rate of reaction is given by

rp = k f2 C XY k b2 C P C X (1.1)

C X0 = C X + C XY
Si
Since enzyme is
i preserved
d

(1.2)

S b tit ti equation
Substituting ti (1.2)
(1 2) in
i (1.1)
(1 1) we gett

rp = (k f2 + k b2 C P )C XY k b2 C P C X0 (1.3)

Assuming the first reversible reaction is in equilibrium gives

kf1
C XY = CXCY (1.4)
k b1
Substituting equation (5) in equation (3) for CX and rearranging for CXY yields

CX0 CS (1.5)
CXY =
k b1
CS
k f1
Substituting equation (6) into equation (2) gives

k b2 k b1 (1.6)

k f2 C X0 C Y C P
k f2 k f1
rp =
k b1
+ CS
k f1
Equation (7) is in Michaelis-Menten form of equation, where

rMax = k f2 C XO
and
k b1
KM =
k f1
Problem 2

Determine
D t i the
th initial
i iti l rate
t off product
d t (P) formation
f ti from
f enzyme X andd
substrate Y that form the complex XY. The reaction kinetics is given by

Solution : We can write the mass balance for the intermediates and product as:

d[(XY )1 ] (2.1)
= k b1 [X ][Y ] (k f1 + k 2 )[(XY )1 ]
dt
d[(XY )2 ]
= k 2 [(XY )1 ] (k f3 )[(XY )2 ] (2.2)
dt
d[P ]
= (k f3 )[(XY )2 ] (2.3)
dt
k f2
where k2 =
k b2
Assuming [(XY)1] and [(XY)2] are in steady state, we can eliminate
[(XY)2] from equation (2.4)
 k f1 [X ][Y ]
[(XY )1 ] = (2.4)
k b1 + k 2

[(XY )2 ] = k2
[(XY )1 ] = k 2 k 1 [X][Y] ( )
(2.5)
k f3 k f3 k b1 + k 2

Substituting equation (2.6) into equation (2.4) we get

d[P] k 2 k f1 [X ][Y ]
rp = = k f3 (2 6)
(2.6)
dt k f3 k b1 + k 2
Since the enzyme is preserved
[X 0 ] = [X] + [(XY )1 ] + [(XY )2 ] (2.7)
Substituting for [X] in terms of [X0] in equation (2.8) we get

k 2 k f3
[X][Y]
(k 2 + k f3 )
rp = (2.8)
k f3 k b1 + k 2
+ [Y ]
k 2 + k f3f k f1f

Hence the KM and Vmax values are reduced by the ratio kf3/(k2+ kf3),
resulting from the presence of the second intermediate [(XY)2].
]
Problem3
The loading of O2 to Hb (hemoglobin) follows a cooperative binding
and is given by

Develop a graphical method for determination of the coefficient n

from measurements of Hb (O2)n

Solution:
Total hemoglobin concentration is given by
[Hb]T = [Hb] + [Hb(O 2 )n ] = Constant

[Hb(O 2 )n ] = [Hb(O 2 )n ]
[Hb]T [Hb] + [Hb(O 2 )n ]
(3.1)
Given reaction is

Hb + nO 2 Hb(O 2 )n
Assuming steady state, therefore
K 1 [Hb][O 2 ] = K 2 [Hb(O 2 )n ] (3.2)
n

From equation (3.1) and equation (3.2) we get

K1
[Hb][O 2 ]n
[Hb(O 2 )n ] = K 2 (3.3)
[Hb]T [Hb] + K 1 [Hb][O 2 ]n
K2
As concentration is directlyy proportional
p p to ppressure,, we can write equation
q
(3.3) as
K1 n n
PO 2
[Hb(O 2 )n ] = K 2 (3.4)
[Hb]T K
1 + 1 n POn 2
K2
After the algebraic manipulations

[Hb(O 2 )n ] = POn 2 (3.5)

[Hb]T K1
+ P n
O
K 2 n 2

Now, binding of oxygen to hemoglobin in terms of partial pressure of

oxygen is given by Hill Equation.

Hill Equation:
POn2
SO2 = (3.6)
P +Pn
50
n
O2
Therefore

SO2 =
[Hb(O 2 )n ]
(3 7)
(3.7)
[Hb]T
From equation (3.6) and equation (3.7)
POn 2
SO 2 = (3.8)
K1
+ P n
O2
K 2 n
Rearranging we get

S O 2 (3 9)
(3.9)
K1
=
n
PO2
1 S O 2
n
K 2
Taking log on both side, we get

S O 2 K1
ln = ln
K n + n ln PO 2
1 S O 2 2 (3 10)
(3.10)
 S O 2
Thus a logarithmic plot of versus PO2 will give a straight line of
slope n 1 S O 2

Fig. 3.1 Binding of oxygen to hemoglobin versus Partial pressure of oxygen

Problem 4

An inhibitor Y is added to the enzymatic reaction at a level of 1.2 gm/L. Their data
were obtained for KM=10 gm/L.

Rate Substrate
1.8 20
1.3 9.8
0.98 6.7
0.8 5.1
0.67 4.2
0.6 3.2
0.45 2.6

a. Identify
Id tif ththe ttype off iinhibition
hibiti (i(i.e. C
Competitive,
titi N Non-competitive
titi or
Uncompetitive)

b Find the Ki (Inhibitor kinetic constant)

b.
Solution: Lets us assume that the given enzymatic reaction is
inhibited by competitive inhibition.
inhibition For an instance of competitive
inhibition the Lineweaver-Burke equation is
1 K M 1 [I] 1
= 1 + + (4 1)
(4.1)
V VMax [S] K i VMax
1 1
So we pplot versus to gget the values of VMax and Ki
V S

Fig. 4.1. 1/V versus 1/S plot

From fig.4.1 we get Vmax=1.8181. Slope = 2.33826, therefore

1 [I]
1 + = 2.3383
VMax Ki
Hence inhibitor constant Ki = -2.087

For Competitive inhibition, Ki is the dissociation constant for the

enzyme inhibitor complex. A lower value of Ki denotes a stronger
inhibition. So as the value of Ki (-2.087) is negative our assumption
was correct and it is a competitive inhibition.

It is important to note that competitive inhibition can be overcome by

adding additional substrate.