Chapter 3

Problem 3.1

S E k2
k1
 ES1 k3
k4
(ES)2 
k5
P  E
rate  k5  ES2

a.) Equilibrium approach

* r values are the same as k values all are rate constants

r1 S E   r2  ES1 (1)

r3  ES1  r4  ES2 (2)
 E0    E    ES1   ES2 (3)
r
from (2)  ES1  4  ES2 (4)
r3
r2 r4  ES2
from (1) + (4)  E   (5)
r1r3 S

Substitute (4) and (5) into (3):

 r2 r4  r1 r4  S  r1 r3  S  
 E 0      ES2
 r1 r3  S 
r1 r3  E 0  S
  ES2 
r2 r4  r1 r4  S   r1 r3 S 
r5 E 0 S r2 r
rate  where Km1  , Km 2  4
Km 2  Km1  S  S r1 r3

b.) Quasi–steady–state approach

d  ES1
 r1  E  S  r2  ES1  r4  ES2  r3  ES 1  0 (6)
dt
d  ES2
 r3  ES1  r4  ES2  r5  ES 2  0 (7)
dt
r4  r5
from (7)  ES1   ES2 (8)
r3

r r r r r r 
from (6) + (8):  E    2 4 2 5 3 5   ES2 (9)
 r1 r3  S 

substitute (8) and (9) into (3):

1
  r2 r4  r2 r5  r3 r5  r1 r4 S   r1 r5 S   r1 r3 S  
 E 0      ES2
 r1 r3  S  
 r1 r3  E 0  S 
 ES2   
 r2 r4  r2 r5  r3 r5  r1 r4  S   r1 r5  S   r1 r3 S  
r5 E0 S
 rate 
 Km2  r5 r3  Km1  S  r5 r1  S
r r
where Km1  2 and Km 2  4
r1 r3

Problem 3.2
* r values are the same as k values all are rate constants
E S r1
r1
 ES r2
r2
EP
d  ES
 r1  E  S  r2  E  P   r1  ES  r2  ES  0 (1)
dt
d  P
rate   r2  ES  r2  E  P  (2)
dt
 E0    E    ES ,  E    E0    ES (3)

r1  r2
from (1):  E    ES (4)
r1 S  r2  P 

 r  S  r2  P   r1  r2 
Combine (3) + (4):  E 0    1   ES
 r1  S   r2  P  
E 0  r1  S  r2  P  
  ES  (5)
r1  S  r2  P   r1  r2

Substitute (3) and (5) into (2):

r1 r2 E 0  S  r2 r2 E 0  P 
rate   r  2  E 0   ES   P 
r1  r2  r1  S  r2  P 

Substituting (ES) in terms of E0:

r1 r2 E  S  r1 r2 E 0  P 
rate 
r1  r2  r1  S  r2  P 

LetVS = r2 E0 and VP = r-1 E0

r1 Vs  S  r2 VP  P 
rate 
r1  r2  r1  S  r2  P 

2
 Divide top and bottom by (r-1 + r2) and let
1 r1 1 r
 and  2
K m r1  r2
1
K P r1  r2

 rate 
V s K1m  S   VP K P  P
S P
1 1 
K m KP

Problem 3.3

k 1  k 2
a) Km   4.5 105 M
k1

b) E 0  106 M
S  103 M
Vm  S
V
K1m  S
but Vm  r2 E0
 V  9.58 104 Msec1

Problem 3.4.

At low substrate concentrations So< 150 mg/l substrate inhibition is negligible.

V0 = Vm0 So / (Km + So) or 1/V0 = 1/ Vm + Km/Vm (1/S0)
For S0 < 150 mg/l Plot 1/V0 versus 1/S0
Slope = Km/Vm0 = 13.8 y-intercept = 1/ Vm = 0.023
Then, Vm = 43.5 mg/l-h and Km = 600 mg/l

At high substrate concentrations above 150 mg/l , substrate inhibition is significant.

V0 = Vm0/ (1+ S0/Ksı) or 1/V0 = 1/Vm0 + S0/Vm Ksı
For S0 > 150 mg/l plot 1/V0 versus S0
Slope = 1/ Vm0Ksı = 2.59x 10-3 Then, Ksı = 8.9 mg/l

Low Ksı indicates severe substrate inhibition.

Problem 3.5.

Plot 1/V versus 1/S at different inhibitor concentrations

Since the lines intercept at the same point on y-axis inhibition is competitive (Constant Vm,
increased Km).
For I = 0 , No inhibitor : From the intercept on y axis , 1/Vm = 0.2 and Vm = 5 mM/h
And from the intercept on X-axis, - 1/Km = -1.2 and Km = 0.83 mM
From 1/V versus 1/S plot for I = 1.3 mM and S0 = 0.50 mM V = 1.3 mM/h
Then, V = Vm S/ (Km(1+I/Ksı)) + S , 1.3 = 5(0.5)/ (0.83(1+1.3/Kı) + 0.5 )
Then Kı = 1.82 mM

3
Problem 3.6 .

a. V = Vm (1 + A/KA) S /(Ks + S) = Vm (1+A/KA) / (1+Ks/S)

Define Vm(1+ A/KA) = Vapp and plot Vapp versus activator (A or M0) concentration
Slope = 9.2x 10-3 = Vm/KA and y –intercept = Vm = 0.04 ml/h
Then, KA = 4.35 ug/l
b. V= Vm (1+ KA) = 0.04(1+60/4.35) = 0.59 ml/h

Problem 3.7.

a. V = KL (S0 - Ss) = Vm Ss / (Km+ Ss)

Ss = S0 – V/KL Calculate Ss at different RPMs
RPM 25 50 100 200 300 400
Ss(mg/l) 33.3 50 78.8 101.2 150 180
b. V = Vm Ss/ (Km+ Ss) or 1/V = 1/Vm + Km/Vm (1/Ss)
Plot 1/V versus 1/Ss at different RPMs
1/Ss 0.03 0.02 0.0127 0.0099 0.0067 0.005
1/V 0.033 0.0277 0.025 0.024 0.0227 0.022
y- intercept: 1/Vm = 0.02 Vm = 50 mg/l
Slope : Km/Vm = 0.42 Then, Km = 21 mg/l

Problem 3.8

H 2 O  CO 2 HCO3  H 
E

Plot 1/V versus 1/S (Lineweaver – Burk plot)

1 1 1
y – intercept =  , x – intercept =
Vm k 2 E 0 Km

1 40
Hydration:   4 103
V  Co2 

1 V  2.50 104 M sec 1

 4000M 1 sec  m
Vm r2  8.93 104 sec 1
1 4000
  100M 1  K m  0.01M
Km 40

1 163.15
Dehydration:   1.31104
V  HCO3 


4
 1 V  7.61105 M sec1
 1.31104 M 1 sec  m
Vm r2  2.72 104 sec1
1 1.31104 1
 M  K m  1.24 102 M
Km 163.15

Problem 3.9

1 1
a.) Plot 1/V versus 1/S:  9.525  0.60
V S
1 0.60
  0.063M1  K m,app  16.0gS / L
K m,app 9.525
SinceKm,app> Km the inhibitor is competitive.

b.)
  I 
K m,app  K m 1  
 KI 

KI 
 I  1.37g I L
K m,app K m  1

5
Problem 3.10

Plot 1/V versus 1/S

a.) For E0 = 1.6 g/L

Km = 0.0246 mmol/ml at 30°C
Km = 0.0238 mmol/mlat 49.6°C

1 mmol
b.) Vm   3.31
0.302 ml.min

c.) The inhibitor is competitive.

  I   I   0.6 mmol mL
K m,app  K m 1  
 KI  K m,app  0.052 mmol mL

Problem 3.11

MW ATPase  5 104 g mol, K m  1104 mol L

molecules ATP
S  0.02 mol L, r2  1104
min molecule enzyme

ATP   ADP  Pi E S ES  E  P

ATPase r1 r2
or r1

Enzyme inactivation kinetics: E = E0e-rt, r = 0.1 min-1

V S
Michaelis-Menten Kinetics: V  m
Km  S

But S Km ,  V  Vm  r2 E

dp
  r2 E 0 e  rt
dt

6
 0.002 


0
dp  r2 E 0  e  rt dt
0

r2 E 0
0.002 M   1 , E 0  2.0 108 M
r
 g   6 g 
 1103 L    5 104
mol
2.0 108   110   1.0g
L  mol   g 
1.0 g
Fraction of pure enzyme   0.1
10 g

 10% of the crude protein was ATPase.

Problem 3.12

At steady – state: reaction rate = mass transfer rate

Vm1 Ss 
V  rL Sb   Ss   J
S 
2

K m  Ss   s
KSI

Solving graphically,

a.) For [Sb] when the enzyme is inhibited and kL has an intermediate value, multiple steady
states are possible.

Multiple steady – states occur as the enzyme changes from being reaction controlled to
being diffusionally controlled.

b.) Yes. Diffusional limitations can decrease the substrate concentration such that it is no
longer inhibitory. Thus the apparent reaction rate will be greater than the intrinsic reaction
rate for [SS] less than [Sb].

7
Problem 3.13

By inspection,Vmax = 125 μmol/min, Km = 20 μmol/L

Plot V versus V/[S](Eadie- Hofstee) where y-int = Vm, x-int. = Vm/Km and slope = - Km.

Plotting the date and using the data points for high substrate concentration,
Vm = 130 μmol/min, Km = 8.24 μmol/L

From the plot it is obvious that the data do not fit into Michaelis – Menten kinetics at low
substrate concentrations.

Some of the error may be attributed to the method since both axes contain v. However, there
are two possible explanations for the deviations from Michaelis – Menten Kinetics:

(i) the enzyme is immobilized, thus diffusional effects become important

(ii) there might be unspecific binding of substrate to the enzyme thus requiring a critical
substrate concentration for the reaction to follow Michaelis – Menten Kinetics.

Problem 3.14

a.) Plot [P] versus t data for both cases.

8
Obtain rates by taking tangents at specific time points.

d  P
v  slope of tangent
dt

Obtain the amount of substrate present for both cases by a mass balance.

H2O  Starch 
 dextrose
MW 18 180

amount of dextrose formed (0.1) = amount of H2O used

(180) (0.1) = 18

 starch remaining = S0 – (dextrose formed – H2O used)

= S0 – 0.9 ∆ P

Using initial time data, a plot of 1/V versus 1/S can be made.

9
Soluble:

1
 0.16
Vm
 1 mg   1 
Vm    
 0.16 mL  min   11600 units 
mg
 5.39 104
mL  min unit enzyme
1 mg
Km   575
0.00174 mL

Immobilized:

1  1 mg   1  4 mg
 0.12, Vm      1.80 10
Vm  0.12 mL  min   46 400 units  mL  min  unit of enzyme
1
Km   575 mg mL
0.00174

b.) Plot In Vm versus 1/T for both cases.

Vm  r2 E 0  E 0 Ae  Ea RT
Ea Ea
ln Vm  ln E 0  ln A   slope 
RT R

Soluble:lnVm = 41.18 – 8299 (1/T)

 cal  Kcal
 E a  8299K 1.987   16.5
 mol  K  mol

10
Immobilized: lnVm = 35.65 – 6582 (1/T)
 cal  Kcal
E a  6582 K 1.987   13.1
 mol  K  mol

c.) Since Km (soluble) = Km (apparent) there is no diffusional limitation.

Problem 3.15

In vitro batch reactors represent a closed system of constant volume, thus Michaelis-Menten
Kinetics and the quasi-steady-state approximation will not describe the system when E0 ≈ S0.
However, intracellular enzyme reactions are open systems where there is a continuous supply
and depletion of substrate and product provided by the interaction of cellular compartments
and the intracellular and extracellular environments. E0 and S0 may be the same, but the
concentrations may be orders of magnitude different either in different organelles or inside
versus outside the cell.

Problem 3.16

Harry’s reasoning is wrong. The soluble enzyme is reaction controlled while the immobilized
enzyme may experience diffusional limitations. Thus the substrate is consumed more slowly
giving rise to a larger apparent half-life. The large particle size may result in an unused or
“reserved” catalytic capacity.

Problem 3.17

(a) Plot of V us. S indicates that this is substrate inhibition

11
(b) Vm andK'm are determined from low substrate concentration where inhibition is not
ineffect:

1 Vm is at x  0  1 Vm  0.049333
Vm  20.3 mg l  h

1 K 'm is at y  0  0  0.049333  1.5355  1 K 'm 

0.049333 1
 ' K 'm  31.1 mg l
1.5355 Km

K 'm
E S ES 
r2
 EP

S
 K SI
ES2
Vm S
u
S
2

K  S
'
m 
K SI

du
Determine KSI from maximum reaction rate; which is determined by setting 0
d S
Smax  K'm KSI

From plot, Smax  90 mg 1  90 mg l   31.1 mg 1 K SI

K SI  260.5 mg 1
Km
1
S

12
(c) Rate of reaction at S = 70 mg/l?

Since inhibition effect is observed at this substrate, we must use (eg) 3.34

Vm S  20.3 70 
u 
S  70 
2 2

K 'm  S   31.1   70  

K SI 260.5
u  11.9 mg l  hr

Problem 3.18

reaction rate with diffusion limitation

Effectiveness factor =
reaction rate with no diffusion limitation

We are given the reaction rate with diffusion limitation. To determine the reaction rate with
no diffusion limitation, lets look at what happens to the rate as the particle gets smaller, and
diffusional limitations are minimized. Aplot of Rate vs. particle size indicates that the rate
reaches a maximum at small particle size (0.1cm). At this size there is no diffusion limitation,
and the rate is N 200 mg/l hr

(a) Effectiveness factor at

100 mg l  hr
D P  0.5cm   
200 mg l  hr
  50
50 mg l  hr
D P  0.7cm   
200 mg l  hr
  0.25

13
b. Assume negligible film resistance, so Sbulk = Ssurface Determine rate u/o diffusional
limitation from effectiveness factor:

rate at D P  0.5cm
rate  w o diff  
 at D P  0.5cm

Plot 1/V vs 1/S to obtain the following:

S0 10 25 50 100 200 250

V 10 20 30 40 45 46
Vno diff 20 40 60 80 90 92

1/ Vm is at x  0 : y  0.0084626  Vm  118.2 mg uA / l  h
–1/Km is at y = 0: 0 =0.0084626 + 0.41489 (–1/Km)
0.0084626 1

0.41489 Km
K m  49.0 mg uA l

Problem 3.19 a

Vm S0
rS   (1)
K S  S0
Vm
R (2)
K S  De
3  (3)

14
 Vm  0.5 
From Equ 1. 10   ; Vm  14 (1)
 0.2  0.5

Vm
From Equ 2.   0.1  1.04 Vm (2)
 0.2 1.5 105  3600 

Multiply (1) × (2) =  Vm  14.55 Vm

since   3 Vm  23.5mmoles L  h
14
  0.595 0.6
23.5
  3 0.595  5.04

b.
Vm
similarly, Vm  14;   0.2
 0.2  1.5 105   3600 
Vm  26.95 Vm   1.925 Vm

Since ϕ η ≈ 3, then, Vm ≈ 80.7 mmoles/L·h

η = 14/80.7  0.173; ϕ = 3/η = 17.3

Problem 3.20

(a) Plot 1/V vs 1/S for all cases to determine the type of inhibition

15
Vmax is the same, regardless of inhibition, but as I increases, –1/Km decreases (apparent), that
is an increased value of Km,app, resulting in a reduction in reaction rate. This is characteristic
of Competitive Inhibition

(b) Determine Vm, Km, KI

Vm, Km can be determined from the I = 0 case.@ I = 0 (1/V) = 0.15782 + 0.19404 (1/S)

1 Vm at 1 S  0 : 1 Vm  0.15782
Vm  6.34 mM h

1/ K m at 1/ Vm   0 : 0  0.15782  0.19404  1/ K m 

0.15782 1
 K m  1.23mM
0.19404 K m

KI must be determined from the I > 0 case

I  1.26 mM  '
 determine K m, app
I  1.95 mM 

I  1.26mM : 1 V   0.15917  0.3098 1 S 

0  0.15917  0.3098  1 K m,app 
0.15917 1
  K m,app  1.95 mM
0.3098 K m,app

16
I  1.95 mM : 1 V   0.15343  0.40383 1 S 
0  0.15343  0.40383  1 K m,app 
0.15343 1
  K m,app  2.63mM
0.40383 K m,app

  I 
K m,app  K m 1  
 KI 

 1.26mM 
I  1.26 mM : 1.95 mM  1.23mM 1  
 KI 
1.26
1.58  1 
KI
1.26
0.58   K I  2.16 mM
KI

 1.95mM 
I  1.95 mM : 2.63mM  1.23mM 1  
 KI 
1.95
2.14  1   K I  1.71mM
KI

We would expect that KI is the same at different concentrations of inhibitor. The differences
in the two values shown here are due to experimental error.

Problem 3.21

K S  200 mg L  0.2 mg cm3

VmSS
K L  Sb  S3  
K m  SS
K L  2.103 m sec  0.2 cm sec
Sb  1000 mg L  1mg cm3

Then

0.1 SS
0.2 1  SS  
0.2  SS
SS2  0.3SS  0.2  0
SS  0.62 mg cm3  620 mg L

b.

17
 Vm SS
V  K L  Sb  SS 
K S  SS
0.1 0.62 
V  0.2 1  0.62   0.076 mg cm 2 sec.
 0.2  0.62 
Problem 3.22

a) Use the graphical technique:

Given

where A is a Function of surface concentration and is determined through rxn Kinetics.

SB = 100 mg/l , KL = 4 x 10 -5 cm/sec

use V  J  KL SB  SS  ; V  KLSB  KLSS ; y  mx  b

where y  V; K L  SB  b; SS  X and  K L  slope

S1  20 mg L V  3.2 106 mg cm 2  s

b)
 .005mg 
S2  rate of supply  rate of diffusion away  V  K L  S2 surface  
 cm3 
K m  5e 3 mg cm3 ; Vm  4e 6 mg km 2  s
Vm S2 surface 
 3.2 106 mg cm 2  s  4e 5 cm s  S2 surface  5e 3 mg cm3 
K m  S2 surface 
S2 surface  .013mg cm3  13mg L
dP
 2.89 106 mg cm 2  s
dt

Problem 3.23 a

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Make total reaction rate curve →Vtot = 2.2.10 – 5 mg/cm2 S

observed rate 2.2 105 mg cm2 S

b)     0.71
rate if no diffusional limitation 3.08 105 mg cm2 S

1.25 105 mg cm S
2

c) P2 P1   1.25 ratio of production rates

1.0 105 mg cm 2 S

d) Sbulk = 370 mg/L

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