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2-10 Enzyme Kinetics

kmole/m
3
s. Care should be taken for the consistency of unit when enzyme
concentration is not expressed in molar unit.
The Michaelis-Menten equation is analogous to the Langmuir isotherm
equation

A
A
C
K C

+
(2.13)
where is the fraction of the solid surface covered by gas molecules and K is
the reciprocal of the adsorption equilibrium constant.
2.2.2. Briggs-Haldane Approach
Again, from the mechanism described by Eqs. (2.5) Eq. (2.6), the rates of
product formation and of substrate consumption are

3
P
ES
dC
k C
dt
(2. 14)

1 2
S
S E ES
dC
k C C k C
dt
(2.15)
Assume that the change of C
ES
with time, dC
ES
/dt, is negligible compared to
that of C
P
or C
S
.

1 2 3
0
ES
S E ES ES
dC
k C C k C k C
dt
(2.16)
Substitution of Eq. (2.16) into Eq. (2.15) confirms that the rate of product
formation and that of the substrate consumption are the same, that is,

3
P S
ES
dC dC
r k C
dt dt
(2.7)
Again, if we assume that the total enzyme contents are conserved,

0
E E ES
C C C + (2.9)
Substituting Eq. (2.9) into Eq. (2.16) for C
E
, and rearranging for C
ES


0
2 3
1
E S
ES
S
C C
C
k k
C
k

+
+
(2.17)
Substitution of Eq. (2.17) into Eq. (2.14) results

0
3
max
2 3
1
E S
P S S
M S
S
k C C
dC dC r C
r
k k
dt dt K C
C
k

+
+
+
(2.18)
which is the same as the Michaelis-Menten equation, Eq. (2.11), except that
the meaning of K
M
is different. In the Michaelis-Menten approach, K
M
is
Enzyme Kinetics 2-11
equal to the dissociation constant k
2
/k
1
, while in the Briggs-Haldane
approach, it is equal to ( k
2
+ k
3
)/k
1
. Eq. (2.18) can be simplified to Eq. (2.11)
if k
2!
k
3
, which means that the product-releasing step is much slower than
the enzyme-substrate complex dissociation step. This is true with many
enzyme reactions. Since the formation of the complex involves only weak
interactions, it is likely that the rate of dissociation of the complex will be
rapid. The breakdown of the complex to yield products will involve the
making and breaking of chemical bonds, which is much slower than the
enzyme-substrate complex dissociation step.

Example 2.1
When glucose is converted to fructose by glucose isomerase, the slow
product formation step is also reversible as:

1
2
3
4
S E ES
ES P E
k
k
k
k
+
+
"""#
$"""
"""#
$"""

Derive the rate equation by employing (a) the Michaelis-Menten and (b) the
Briggs-Haldane approach. Explain when the rate equation derived by the
Briggs-Haldane approach can be simplified to that derived by the Michaelis-
Menten approach.
Solution:
(a) Michaelis-Menten approach: The rate of product formation is

3 4 P ES P E
r k C k C C (2.19)
Since enzyme is preserved,

0
E E ES
C C C + (2.20)
Substitution of Eq. (2.20) into Eq. (2.19) for C
E
yields

0
3 4 4
( )
P P ES P E
r k k C C k C C + (2.21)
Assuming the first reversible reaction is in equilibrium gives

1
2
ES E S
k
C C C
k
(2.22)
Substituting Eq. (2.22) into Eq. (2.20) for C
E
and rearranging for C
ES

yields
2-12 Enzyme Kinetics

0
2
1
E S
ES
S
C C
C
k
C
k

+
(2.23)
Substituting Eq. (2.23) into Eq. (2.8) gives

0
4 2
3
3 1
2
1
E S P
P
S
k k
k C C C
k k
r
k
C
k
_

+
(2.24)
(b) Briggs-Haldane approach: Assume that the change of the complex
concentration with time, dC
ES
/dt, is negligible. Then,

1 2 3 4
0
ES
S E ES ES P E
dC
k C C k C k C k C C
dt
+ (2.25)
Substituting Eq. (2.20) into Eq. (2.25) for C
E
and rearranging gives

0
1 4
2 3 1 4
( )
( )
E S P
ES
S P
C k C k C
C
k k k C k C
+

+ + +
(2.26)
Inserting Eq. (2.26) into Eq. (2.19) for C
ES
gives

0
4 2
3
3 1
2 3 4
1 1
P
E S P
S P
k k
k C C C
k k
r
k k k
C C
k k
_

+
+ +
(2.27)
If the first step of the reaction, the complex formation step, is much faster
than the second, the product formation step, k
1
and k
2
will be much larger
than k
3
and k
4.
Therefore, in Eq. (2.27),

2 3 2
1 1
k k k
k k
+
(2.28)
and

4
1
0
k
k
(2.29)
which simplifies Eq. (2.27) into Eq. (2.24).

2.2.3. Numerical Solution
From the mechanism described by Eqs. (2.5) and (2.6), three rate equations
can be written for C
P,
C
ES
, and C
S
as
Enzyme Kinetics 2-13

3 ES
P
dC
k C
dt
(2.14)

1 2 3
ES
S E ES ES
dC
k C C k C k C
dt
(2.30)

1 2
S
S E ES
dC
k C C k C
dt
+ (2.31)
Eqs. (2.14), (2.30), and (2.31) with Eq. (2.9) can be solved simultaneously
without simplification. Since the analytical solution of the preceding
simultaneous differential equations are not possible, we need to solve them
numerically by using a computer. Among many software packages that solve
simultaneous differential equations, Advanced Continuous Simulation
Language (ACSL, 1975) is very powerful and easy to use.
The heart of ACSL is the integration operator, INTEG, that is,
R INTEG(X,R0)
implies

0
0
t
R R Xdt +


Original set of differential equations are converted to a set of first-order
equations, and solved directly by integrating. For example, Eq. (2.14) can be
solved by integrating as

0
3
0
t
P P ES
C C k C dt +


which can be written in ACSL as
CP = INTEG(K3*CES,CP0)
For more details of this simulation language, please refer to the ACSL User
Guide (ACSL, 1975).
You can also use Mathematica (Wolfram Research, Inc., Champaign, IL)
or MathCad (MathSoft, Inc., Cambridge, MA). to solve the above problem,
though they are not as powerful as ACSL.
It should be noted that this solution procedure requires the knowledge of
elementary rate constants, k
1,
k
2,
and k
3.
The elementary rate constants can be
measured by the experimental techniques such as pre-steady-state kinetics
and relaxation methods (Bailey and Ollis, pp. 111113, 1986), which are
much more complicated compared to the methods to determine K
M
and r
max
.
Furthermore, the initial molar concentration of an enzyme should be known,
which is also difficult to measure as explained earlier. However, a numerical
2-14 Enzyme Kinetics
solution with the elementary rate constants can provide a more precise picture
of what is occurring during the enzyme reaction, as illustrated in the
following example problem.

Example 2.2
By employing the computer method, show how the concentrations of
substrate, product, and enzyme-substrate complex change with respect to
time in a batch reactor for the enzyme reactions described by Eqs. (2.5) and
(2.6). The initial substrate and enzyme concentrations are 0.1 and 0.01 mol/L,
respectively. The values of the reaction constants are: k
1
= 40 L/mols, k
2
= 5
s
1
, and k
3
= 0.5 s
1
.
Table 2.2
ACSL Program for Example 2.2
PROGRAMENZY- EX2 ACSL
I NI TI AL
ALGORI THMI ALG=5 $ ' RUNGE- KUTTA FOURTH ORDER'
CONSTANT K1=40. , K2=5. , K3=0. 5, CE0=0. 01, . . .
CS0=0. 1, CP0=0. 0, TSTOP=130
CI NTERVAL CI NT=0. 2 $ ' COMMUNI CAI TON I NTERVAL'
NSTEPS NSTP=10
VARI ABLE TI ME=0. 0
END $ ' END OF I NI TI AL'
DYNAMI C
DERI VATI VE
DCSDT=- K1*CS*CE+K2*CES
CS=I NTEG(DCSDT, CS0)
DCESDT=K1*CS*CE- K2*CES- K3*CES
CES=I NTEG(DCESDT, 0. 0)
CE=CE0- CES
DCPDT=K3*CES
CP=I NTEG(DCPDT, CP0)
END $ ' END OF DERI VATI VE SECTI ON'
TERMT(TI ME. GE. TSTOP)
END $ ' END OF DYNAMI C SECTI ON'
END $ ' END OF PROGRAM'

Table 2.3
Executive Command Program for Example 2.2
SET TI TLE =' SOLUTI ON OF EXAMPLE 2. 2'
SET PRN=9
OUTPUT TI ME, CS, CP, CES, ' NCI OUT' =50 $' DEFI NE LI ST TOBE PRI NTED'
PREPAR TI ME, CS, CP, CES $' DEFI NE LI ST TOBE SAVED'
START
SET NPXPPL=50, NPYPPL=60
PLOT ' XAXI S' =TI ME, ' XLO' =0, ' XHI ' =130, CS, CP, CES, ' LO' =0, ' HI ' =0. 1
STOP

Enzyme Kinetics 2-15
Solution:
To determine how the concentrations of the substrate, product, and
enzyme-substrate complex are changing with time, we can solve Eqs. (2.14),
(2.30), and (2.31) with the enzyme conservation equation Eq. (2.9) by using
ACSL.
The ACSL program to solve this problem is shown in Table 2.2, which is
composed of four blocks: PROGRAM, INITIAL, DYNAMIC, and
DERIVATIVE. Each block when present must be terminated with an END
statement. For the integration algorithm (IALG), Runge-Kutta fourth order
(IALG = 5) was selected, which is default if not specified.
5
The calculation

5
Other algorithms are also available for the selection. They are Adams-Moulton
(IALG = 1), Gears Stiff (IALG = 2), Runge-Kutta first order or Euler (IALG = 3),
and Runge-Kutta second order (IALG = 4). The Adams-Moulton and Gear's Stiff
are both variable-step, variable-order integration routines. For the detailed
description of these algorithms, please refer to numerical analysis textbooks, such
Figure 2.4 Solution of Example 2.2 by using MathCad
0 50 100
0
0.05
0.1
Cs
Ces
Cp
t
Cp S
4
: Ces S
3
: Cs S
2
: t S
1
:
Solve using adaptive Runge-Kutta method
S Rkadapt C0 t0 , t1 , N , D , ( ) :
Number of solution values on [t0, t1]
N 13 :
C0
0.1
0
0

,
:
Vector of initial function values
Initial and terminal values of independent variable
t1 130 : t0 0 :
Note:
D t C , ( )
k1 C
1
0.01 C
2
( ) k2 C
2
+
k1 C
1
0.01 C
2
( ) k2 C
2
k3 C
2

k3 C
2

1
1
1
1
]
:
C1 = Cs
C2 = Ces
C3 = Cp
k3 0.5 : k2 5 : k1 40 :
Default origin is 0.
ORIGIN 1 :
2-16 Enzyme Kinetics
interval (integration step size) is equal to the comunication interval (CINT)
divided by the number of steps (NSTP). The run-time control program is
shown in Table 2.3. Figure 2.4 shows the solution by MathCad.

2.2.4. Evaluation of Michaelis-Menten Parameters
In order to estimate the values of the kinetic parameters, we need to make a
series of batch runs with different levels of substrate concentration. Then the
initial reaction rate can be calculated as a function of initial substrate
concentrations. The results can be plotted graphically so that the validity of
the kinetic model can be tested and the values of the kinetic parameters can
be estimated.
The most straightforward way is to plot r against C
S
as shown in Figure
2.2. The asymptote for r will be r
max
and K
M
is equal to C
S
when r = 0.5 r
max
.
However, this is an unsatisfactory plot in estimating r
max
and K
M
because it is
difficult to estimate asymptotes accurately and also difficult to test the
validity of the kinetic model. Therefore, the Michaelis-Menten equation is
usually rearranged so that the results can be plotted as a straight line. Some of
the better known methods are presented here. The Michaelis-Menten
equation, Eq. (2.11), can be rearranged to be expressed in linear form. This
can be achieved in three ways:

max max
S M S
C K C
r r r
+ (2.32)

max max
1 1 1
M
s
K
r r r C
+ (2.33)

max M
S
r
r r K
C
(2.34)
An equation of the form of Eq. (2.32) was given by Langmuir (Carberry,
1976) for the treatment of data from the adsorption of gas on a solid surface.
If the Michaelis-Menten equation is applicable, the Langmuir plot will result
in a straight line, and the slope will be equal to 1/r
max
. The intercept will be
max M
K r , as shown in Figure 2.5.

as Gerald and Wheatley (1989), Chappra and Canale (1988), Carnahan et al.
(1969), and Burden and Faires (1989). }
Enzyme Kinetics 2-17
Similarly, the plot of 1/r versus 1/C
S
will result in a straight line according
to Eq. (2.33), and the slope will be equal to
max M
K r . The intercept will be
1/r
max
, as shown in Figure 2.6. This plot is known as Lineweaver-Burk plot
(Lineweaver and Burk, 1934).
The plot of r versus r/C
S
will result in a straight line with a slope of K
M
and an intercept of r
max
, as shown in Figure 2.7. This plot is known as the
Eadie-Hofstee plot (Eadie, 1942; Hofstee, 1952).
The Lineweaver-Burk plot is more often employed than the other two
plots because it shows the relationship between the independent variable C
S
and the dependent variable r. However, 1/r approaches infinity as C
S
decreases, which gives undue weight to inaccurate measurements made at
low substrate concentrations, and insufficient weight to the more accurate
0 20 40 60 80
5
10
15
20
C
S
r
C
S
K
M
r
max
1
r
max

Figure 2.5 The Langmuir plot (K
M
= 10, r
max
= 5).

0 -0.05 0.05 0.10 0.15
0.2
0.4
0.6
1
r
1
C
S
1
r
max
K
M
r
max

Figure 2.6 The Lineweaver-Burk plot (K
M
= 10, r
max
= 5).