Exercices Advanced Enzymology

Y. Engelborghs

A. Diagnosis for inhibitors:

Four types of reversible inhibitors: competitive, uncompetitive, noncompetitive, mixed. To
identify the type of inhibitor we can construct a Lineweaver-Burk plot (1/v vs. 1/[S] ) for
with varying [I] and interpret the different meaning of the intercept and slope of the plot. For
a simple enzyme-substrate system the interpretation is:

Etot 1 K 1
= + M
v0 kcat kcat [ S ]

Intercept slope Slope/intercept

= 1/kcat KM/kcat KM

1. Competitive inhibition: equilibrium derivation:

Etot = [ E ] + [ ES ] + [ EI ]

Eactive = [ ES ]

[S ]
KM
v0 = vmax
[S ] [ I ]
1+ +
KM KI

Lineweaver Birk plot :

Etot 1 K M [ I ]K M
= ( + + 1)
v0 kcat [ S ] [ S ]K I

Identification of LB-parameters via Lineweaver-Birk plots for different values of [I]:

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 Intercept Slope Slope/intercept
Competitive 1 KM [I ] [I ]
= Ct (1 + ) K M (1 + )
kcat kcat KI KI

2. Uncompetitive inhibition equilibrium derivation:

Etot = [ E ] + [ ES ] + [ ESI ]

Eactive = [ ES ]

[S ]
KM
v0 = vmax
[ S ] [ S ][ I ]
1+ +
KM KM KI

Lineweaver Birk plot :

Etot 1 KM [I ]
= ( + + 1)
v0 kcat [ S ] K I

Identification of LB-parameters:

Intercept Slope Slope/intercept

Uncompetitive 1 [I ] KM [I ]
(1 + ) = Ct K M /(1 + )
kcat KI kcat KI

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 3. Non-competitive inhibition:

Etot = [ E ] + [ ES ] + [ EI ] + [ ESI ]

Eactive = [ ES ]

[S ]
KM
v0 = vmax
[ S ] [ I ] [ S ][ I ]
1+ + +
KM KI KM KI

Lineweaver Birk plot :

Etot 1 K M [ I ]K M [ I ]
= ( + + + 1)
v0 kcat [ S ] [ S ]K I K I

Identification of LB-parameters:

Intercept Slope Slope/intercept

noncompetitive 1
kcat
[I ]
(1 + )
KI
KM
kcat
[I ]
(1 + )
KI
K M = Ct

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 4. Overall Diagnosis of Inhibitors:

Make primary Lineweaver-Birk plots of 1/v vs. 1/[A] for different values of [I]

Diagnosis: follow red diagonal

Intercept Slope Slope/intercept

Competitive 1 KM [I ] [I ]
= Ct (1 + ) K M (1 + )
kcat kcat KI KI
Uncompetitive 1 [I ] KM [I ]
(1 + ) = Ct K M /(1 + )
v max KI kcat KI
noncompetitive 1
kcat
[I ]
(1 + )
KI
KM
kcat
[I ]
(1 + )
KI
K M = Ct
of primary Lineweaver-Birk plot

Logic:

competitive inhibition; Substrate can compete away I at high concentration,

therefore vmax = Ct

uncompetitive: I does not bind E therefore kcat/KM = Ct

(Mnemotechnic: uncompetitive I = I unactive at the unoccupied Enzyme)

Noncompetitive: I binds equally well to E as to ES therefore KM = Ct

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 B. Diagnosis for Two substrate kinetics

With ternary complex :

- random sequence of binding;

- ordered mechanism

Without ternary complex:

- Ping pong mechanism : 2nd substrate binds after first

product formation

- Theorell Chance mechanism: a ternary complex is formed

but it is not populated because the 1st product formation is very fast.

1. Ternary complex formation with random order

Can be described via equilibrium assumption:

Etot = [ E ] + [ EA] + [ EB ] + [ EAB ]

Eactive = [ EAB ]

[ A][ B ]
K AKB f
v0 = vmax
[ A] [ B ] [ A][ B]
1+ + +
K A KB K AKB f

Lineweaver Birk plot :

Etot 1 K AKB f KB f K A f
= ( + + + 1)
v0 kcat [ A][ B ] [ B ] [ A]

f or 1 if A and B enforce each others binding then f<1, for a Briggs-Haldane

enzyme f may be >1.

Diagnosis: make primary Lineweaver-Birk plot with 1/v vs. 1/[A] with varying [B]:
Intercept Slope Slope/intercept
Random sequence 1 fK fK A K
(1 + B ) (1 + B ) K A if f = 1
kcat [ B] kcat [ B]
@Intercept

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 Individual parameters can be obtained from secondary plots of intercepts or slopes vs.
1/[B]

Summary: information from secondary plots

Secondary plot intercept Slope Slope/intercept

Intercept vs 1/[B]-plot 1/kcat fKB/kcat fKB
Slope vs 1/[B]-plot fKA/kcat fKAKB/kcat KB
fKA f
KA

2. Ternary complex formation with ordered sequence (A obligatory

first)

Etot = [ E ] + [ EA] + [ EAB ]

Eactive = [ EAB ]

[ A][ B ]
K AKB
v0 = vmax
[ A] [ A][ B]
1+ +
K A K AKB

Lineweaver Birk plot :

Etot 1 K AKB KB
= ( + + 1)
v0 kcat [ A][ B] [ B]

Parameters of primary LB-plot:

Intercept Slope Slope/intercept

ordered 1 K K A KB K K
(1 + B ) ( ) K A B / 1 + B
v max [B] v max [ B ] [ B] [ B]
@Intercept =0
of primary Lineweaver-Birk plot

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 3. For ping pong (enzyme substitution) mechanism
E + AX
EX + A
EX + B
E + BX

EX is a modified enzyme. The MM equation cannot be derived via equilibrium

assumptions, but has to be derived via transit-times (see notes). The result looks formally
very similar to the previous equation but there is no term (1) for E in the mass balance for
Etot

[ A][ B]
K AKB
v = vmax
[ A] [ B ] [ A][ B]
+ +
K A KB K AKB

Lineweaver Birk plot

Etot 1 KB K A
= ( + + 1)
v0 kcat [ B ] [ A]

Diagnostic: make Lineweaver-Birk plot with respect to [A]at varying

[B]. This leads to:

Intercept Slope Slope/intercept

Ping pong 1 K KA K
(1 + B ) = Ct K A /(1 + B )
kcat [ B] kcat [ B]
Parallel lines

Individual parameters can be obtained from secondary plots.

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 KA
slope = independent of [ B] = primary LB plot : parallel lines
kcat

1 K 1
int ercept = (1 + B ) gives for [ B ]
kcat [ B] kcat

slope KA
= gives K A for [ B]
int ercept (1 + K B )
[ B]

Further proof for ping-pong: demonstrate existence of EX

4. Chance-Theorell: ternary complex is not populated kinetically

Cannot be distinguished from ordered reaction on the basis of initial rate studies

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 5. Overall analysis two-substrate reactions:
Primary Lineweaver-Birkplot of 1/v vs. 1/[A]

Individual parameters can be obtained from secondary plots of intercept or slope vs 1/[B]
@ secondary plot of slope vs. 1/[B]
Intercept Slope Slope/intercept
Random sequence 1 fK fK A K
(1 + B ) (1 + B )
kcat [ B] kcat [ B]
@grows with
positive intercept
ordered 1 K K A KB K K
(1 + B ) ( ) K A B / 1 + B
kcat [ B] kcat [ B] [ B] [ B]
@grows with
intercept =0
Ping pong 1 KB KA K
(1 + ) = Ct K A /(1 + B )
kcat [ B] [ B]
kcat
@ remains ct
Theorell-Chance Similar to ordered

of primary Lineweaver-Birk plot

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C. Exercises

Exercise NR 1 acetylcholinesterase (Price & Dwek, p184)

The Effect of choline (C) on the reaction catalysed by acetylcholine-esterase was studied and
gave the following results: [AC] = acetylcholine concentration

[AC] mM [AC] mM [AC] mM [AC] mM [AC] mM

[C] mM 0.1 0.15 0.25 0.40 0.70

0 28.5@ 37.5 50.0 61.5 73.7

20 11.9 15.6 20.9 25.7 30.7
40 7.5 9.9 13.2 16.2 19.4
@ relative velocity (in arbitrary units)

Task: Analyse the data

Primary Lineweaver-Birk plots

acetylcholinesterase

0.14

0.12

0.10

0.08
1/v

0.06

0.04

0.02

0.00
0 2 4 6 8 10 12

1/[AC] 1/mM
C=0
C=20
C=40

Figure 1: Lineweaver-Birk plot for different [C]

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 1) vmax is not constant but decreases when C increases = not competitive inhibition.
2) Intercepts and slopes increase when [C] increases !!!
3) Slope/intercept= constant = KMAC is independent on C: therefore : non competitive
inhibition

Intercept Slope Slope/intercept

C=0 0,98 x 10-2 2,58 x 10-3 0,263

C=20 2,36x 10-2 5,93 x 10-3 0,251
C=40 3,9x 10-2 9,35 x 10-3 0,24

Conclusion: KM = constant noncompetitive inhibition . Make secondary plots to get

parameters:

Exercise NR2 Fructose bisphosphate (FBP)

Fructose bisphosphatase (reaction catalysed?) is inhibited by AMP. The following data were
obtained from a study of the rat liver enzyme:
uM = micromolar

[FBP] uM [FBP] uM [FBP] uM [FBP] uM [FBP] uM

[AMP] uM 4 6 10 20 40

0 0.059@ 0.076 0.101 0.125 0.150

8 0.034 0.043 0.056 0.071 0.083

@ velocity in katal.kg-1
Analyse these data.

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Exercise NR3 Nucleoside diphosphokinase

The enzyme nucleoside diphosphokinase will catalyse the following reaction:

GTP + dGDP GDP + dGTP in presence of Mg2+

In an experiment with an enzyme isolated from erythrocytes, the following result were
obtained:

[GTP] uM [GTP] uM [GTP] uM [GTP] uM

[dGDP]uM 22 30 50 200

20 0.095 0.112 0.141 0.196

25 0.102@ 0.120 0.155 0.223
40 0.112 0.136 0.180 0.284
100 0.125 0.156 0.218 0.385

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@ velocity in katal.kg-1

What is the likely mechanism for the enzyme? How could you check your conclusion?

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Exercise NR4 Creatine Kinase

A study was made of the reaction catalysed by creatine kinase.

Creatine + ATP phosphocreatine + ADP (in the presence of Mg2+ )

The following data were obtained:

[ATP] mM [ATP] mM [ATP] mM [ATP] mM

0.46 0.62 1.23 3.68
[creatine] mM
6 0.38@ 0.46 0.66 0.97
10 0.55 0.68 0.95 1.31
20 0.85 1.00 1.34 1.80
40 1.18 1.38 1.72 2.29
@ velocity in katal.kg-1

Evaluate the kinetic parameters for this reaction. From other data it appears that the
reaction proceeds via a random order ternary complex mechanism. What can you deduce
about the binding of substrates to the enzyme?

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Exercise Nr 5 Alcohol deydrogenase

The following data were obtained in a study of the reaction catalysed by yeast alcohol
dehydrogenase:

Ethanol + NAD+ acetaldehyde + NADH

[NAD+] mM [NAD+] mM [NAD+] mM [NAD+] mM

[Ethanol] mM 0.05 0.10 0.25 1.0

10 0.30@ 0.51 0.89 1.43

20 0.44 0.75 1.32 2.11
40 0.57 1.00 1.72 2.76
200 0.76 1.31 2.29 3.67
@ velocity in katal.kg-1

Determine the mechanism and the kinetic parameters of this reaction.

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 Exercise NR6
The effect of the products (pyrvate) on the reaction catalysed by rabbit muscle lactate
dehydrogenase was studied, with the results in the following tables.

Reaction: NAD+ + lactate NADH + pyruvate

Experiment 1: Following data were obtained at fixed concentration of [NAD+] = 1,5

mM

[Lact] mM [Lact] mM [Lact] mM [Lact] mM

[Pyruvate] uM 1.5 2.0 3.0 10.0

0 1.88@ 2.36 3.10 5.81

40 1.05 1.34 1.88 4.19
80 0.73 0.94 1.34 3.27
@ velocity in katal.kg-1

Experiment 2: Following data were obtained at fixed concentration of Lactate [Lact] = 15

mM

[NAD+] mM [NAD+] mM [NAD+] mM [NAD+] mM

[Pyruvate] uM 0.5 0.7 1.0 2.0

0 3.33@ 3.91 4.50 5.43

30 2.65 3.13 3.60 4.33
60 1.97 2.30 2.66 3.21
@ velocity in katal.kg-1

What is the interpretation of these data?

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 Exercise NR 7
Chymotrypsin catalyses the hydrolysis of p-Nitrophenolacetate (PNPA) to yield
p-nitrophenol and acetate. The production of p-nitrophenol (PNP) was followed as a
function of time with the following results:

experiment 1: [chymotrypsin]= 23 uM; [PNPA] = 500 uM

t(s) 0 15 30 60 90 120 150 180

[PNP]uM 0 18.3 22.2 29.1 35.7 41.7 48.0 54.3

Experiment 2: chymotrypsin]= 12 uM; [PNPA] = 500 uM

t(s) 0 15 30 60 90 120 150 180
[PNP]uM 0 10.3 12.6 16.2 19.7 23.3 26.8 30.4

What do you deduce from these data?

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