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Journal of Enzyme Inhibition and Medicinal Chemistry, December 2006; 21(6): 617634

Deriving the rate equations for product inhibition patterns in

bisubstrate enzyme reactions
2, DRAGINJA PERIC
IN1, &
VLADIMIR LESKOVAC1, SVETLANA TRIVIC
3

JULIJAN KANDRAC
1

Faculty of Technology, University of Novi Sad, Yugoslavia, 2Faculty of Sciences, University of Novi Sad, Yugoslavia, and
Faculty of Agriculture, University of Novi Sad, Yugoslavia

(Received 17 January 2006; accepted 9 April 2006)

Abstract
In this work, the full rate equations for 17 completely reversible bisubstrate enzyme kinetic mechanisms, with two substrates in
the forward and two in the reverse direction, have been presented; among these are rapid equilibrium, steady-state, and mixed
steady-state and rapid equilibrium mechanisms. From each rate equation eight product inhibition equations were derived,
four for the forward and four for the reverse direction. All the corresponding product inhibition equations were derived in full;
thus a total of 17 8 136 equations, were presented. From these equations a list of product inhibition patterns were
constructed and presented in a tabular form, both for the primary plots (intercept effects) and the secondary plots (slope
effects).
The purpose of this work is to help investigators in practical work, especially biologists working with enzymes, to choose
quickly an appropriate product inhibition pattern for the identification of the kinetic mechanism. The practical application of
above product inhibition analysis was illustrated with three examples of yeast alcohol dehydrogenase-catalyzed reactions.

Introduction
The majority of enzyme-catalyzed reactions in nature
are bisubstrate reactions with two substrates and two
products of reaction; monosubstrate reactions are rare,
and trisubstrate reactions are far less numerous then
bisubstrate (Enzyme nomenclature 1992; [11,16]). [6]
and [2 4] have pioneered the derivation of rate
equations for several major bisubstrate mechanisms,
which was followed by other authors [16]. Several rate
equations for major sequential bisubstrate mechanisms are presented in extenso in a textbook format by
[17] and by [14] and in a review format in several
volumes of Methods in Enzymology. However, the
textbook and the review format published so far
present only a fraction of all possible bisubstrate
mechanisms that occur in nature.
The purpose of this article is: (a) To review the
methods for the derivation of full rate equations for

bisubstrate reactions and derive equations for most

mechanisms that occur in nature. (b) Further, to
derive all possible product inhibition equations in the
double reciprocal form from the full rate equations.
(c) Finally, the aim is to use the above product
inhibition equations to construct the product inhibition patterns in a tabular form. In this way, the
product inhibition tables can help the students in
practical work with enzymes and help them to identify
an appropriate kinetic mechanism in question. In
order to learn how this is done in practical kinetics,
students need to work some examples with real kinetic
data.
Bisubstrate enzyme reactions
Bisubstrate enzyme reactions, with two substrates
(A and B) and two products of the reaction (P and Q),
can be classified into two broad categories: rapid

Correspondence: V. Leskovac, Faculty of Technology, Bulevar Cara Lazara 1, 21000 Novi Sad, Yugoslavia. E-mail: [email protected]
ISSN 1475-6366 print/ISSN 1475-6374 online q 2006 Informa UK Ltd.
DOI: 10.1080/14756360600829381

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618 V. Leskovac et al.

equilibrium Bi Bi and steady-state Bi Bi reactions. The
derivation of rate equations for each category is very
different and will be further treated in separate
sections. In bisubstrate reactions, the designation of A,
B, and P, Q are arbitrary; however, when dealing with
NAD(P)-dependent dehydrogenases, it is customary
to assign A to oxidized coenzyme and B to alcohol
substrate in the forward direction, and P to carbonyl
substrate and Q to reduced coenzyme in the reverse
direction. This choice is not voluntary, but makes an
important difference when one is dealing with tables of
product inhibition patterns (Tables II V).

Rapid equilibrium Bi Bi mechanisms

It is important to realize that in rapid equilibrium
mechanisms all forms of enzyme are in a rapid
thermodynamic equilibrium, which is established
immediately after mixing the substrates with the
enzyme. For rapid equilibrium systems, the rate
equations can be obtained easily; no derivation is
really necessary, and the rate equations can be written
directly from an inspection of the equilibriums
between the enzyme species [14]. The full rate
equations for all rapid equilibrium Bi Bi mechanisms
are easily derived from the general rate equation for the
Rapid equilibrium random Bi Bi mechanism (Scheme
1) with EAP and EBQ complexes (Equation I).

Table I. Survey of rapid equilibrium Bi Bi mechanisms.

Mechanism

Omission of respective
enzyme form from Scheme 1,
or respective denominator
term from Equation (I)

Delete EAP and EBQ

RE Random Bi Bi without
dead-end complexes
2 RE Random Bi Bi with
EAP complex
3 RE Random Bi Bi with
EBQ complex
4 RE Random Bi Bi with
EAP and EBQ complexes
5 RE Random Bi Bi with
EAP, EBQ, PE, and PEP
complexes
6 RE Ordered Bi Bi without
dead-end complexes
7 RE Ordered Bi Bi with
dead-end EAP complex
8 RE Ordered Bi Bi with
dead-end EBQ complex
9 Forward RE random, reverse
RE ordered Bi Bi
10 Forward RE ordered, reverse
RE random Bi Bi
11 RE ordered Bi Bi, with QE
and QEQ complex

Delete EBQ
Delete EAP
No deletion
Special case

Delete EB, EP, EAP,

and EBQ
Delete EB, EP, and EBQ
Delete EB, EP, and EAP
Delete EP, EAP, and EBQ
Delete EB, EAP, and EBQ
Special case

Scheme 1.

In Scheme 1 all kinetic constants represent true

dissociation constants of the respective enzyme forms.
Summary of Rapid equilibrium Bi Bi mechanisms and
rate equations
In this article, we shall describe 11 rapid equilibrium
Bi Bi mechanisms which are listed in Table I. Each
mechanism can be easily written down schematically

simply by omitting from Scheme 1 the enzyme form

indicated in Table I. Further, the full rate equation
for each mechanism can be easily written down from
Equation (I), simply by omitting from its denominator the terms indicated in Table I. The rapid
equilibrium mechanisms always contain one denominator term for each enzyme form in the mechanism.
The numerator term is always the same in all
mechanisms.

Deriving the rate equations for product inhibition

Special cases in Rapid equilibrium Bi Bi mechanisms

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There are two special cases in Rapid equilibrium Bi Bi

mechanisms. The first is the Rapid equilibrium random
Bi Bi with EAP, EBQ, PE, and PEP complexes.

Mechanism 5 (Scheme 2).

619

In this ordered mechanism, the complexes EB, EP,

EAP, and EBQ do not form, but a substrate Q binds
twice to the enzyme, once in its physiological position
and the second time in a nonphysiological position, so
the complexes QE and QEQ are formed. The full rate
equation (Equation III) is obtained simply by adding
the terms Q/KX Q 2/aKXKiQ into the denominator

Scheme 2.

In this mechanism, a substrate P binds twice to

the enzyme, once in its physiological position and
the second time in a nonphysiological position, so
the complexes PE and PEP are formed. Note that
complex EP is different from the complex PE. The
full rate equation (Equation II) is obtained by
adding the terms P/KX P 2/aKXKiP into the
denominator of Equation (I). In Equation (II),
two new dissociation constants appear, KX and
aKX.
The second special case is the Rapid equilibrium
ordered Bi Bi mechanism with QE and QEQ
complexes.

Mechanism 11 (Scheme 3).

Scheme 3.

of Equation (I), and by omitting the terms for EB, EP,

EAP, and EBQ.
Derivation of rate equations in the double
reciprocal form
The purpose of this work was to derive the product
inhibition equations from rate equations in the double
reciprocal form, for major sequential bisubstrate
mechanisms. The derivation of rate equations in the
double reciprocal form is really simple. Take for
example the Rapid equilibrium random mechanism
with EAP and EBQ complexes (Mechanism 4 in
Table I). One takes the inverse form of the full rate
equation (Equation I) and calculates the inverse of the
initial rate (1/vo) for A in the presence of B and Q, for
B in the presence of A and Q, for A in the presence of
B and P, and so on; in doing so, the terms with the
missing substrate are always deleted. Note that V2 in
Equation (I) is zero if only A, B, and Q are present,
as well as if only A, B, and P are present; the same is
true for V1 in the reverse direction. Since there are two
substrates and two products of reaction, we obtain
four product inhibition equations in the forward and
four in the reverse direction (Equations 25 32).
From the full rate equation, for each of 11
mechanisms, eight product inhibition equations were
derived in the double reciprocal form, a total of
11 8 88 equations, and presented below
(Equations 188).

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620 V. Leskovac et al.

Rapid equilibrium random Bi Bi without dead-end
complexes


1
1
KB

1
A:
vo V 1
B



KA
K iB
Q
1
1
1
1

K iQ A
V1
B

B:



1
1
KA

1
vo V 1
A



KB
K iA
Q
1
1

1
K iQ B
V1
A

C:

C:

D:

E:

F:

G:

H:

vo

Rapid equilibrium random Bi Bi with dead-end EAP

complex


1
1
KB

1
A:
vo V 1
B



KA
K iB
Q
1
1
1

9
K iQ A
V1
B

B:
2

V1

AB
K iA K B



1
1
KA

1
vo V 1
A



KB
K iA
Q
1
1

1
K iQ B
V1
A

2 V2

PQ
K P K iQ

AP
P
P
1 KAiA KBiB KPiP KQiQ K AB
K PQ
K iBBQ
K iiQ K iA K iiP K X aK X K iP
iA K B
P K iQ



1
1
KB

1
vo V 1
B



KA
K iB
P
1
1

1
K iP A
V1
B


1
1
KA

1
vo V 1
A



KB
K iA
P
1
1
1

K iP B
V1
A


1
1
KP

1
vo V 2
P



KQ
K iP
A
1
1
1

K iA Q
V2
P


1
1
KQ

1
vo V 2
Q



KP
K iQ
A
1
1
1

K iA P
V2
Q


1
1
KP

1
vo V 2
P



KQ
K iP
B
1
1

1
K iB Q
V2
P


1
1
KQ

1
vo V 2
Q



KP
K iQ
B
1
1

1
K iB P
V2
Q

10

D:
3

E:

F:

G:

H:

11



1
1
KA

1
vo V1
A




KB
K iA
P
P 1 12
1
1

K iP
K iiP B
V1
A



1
1
KP

1
vo V 2
P




KQ
K iP
A
A 1 13
1
1

K iA
K iiA Q
V2
P



1
1
KQ
A
1

1
vo V 2
K iiA
Q



KP
K iQ
A
1
1

1
K iA P
V2
Q


1
1
KP

1
vo V 2
P



KQ
K iP
B
1
1
1

K iB Q
V2
P


1
1
KQ

1
vo V 2
Q



KP
K iQ
B
1
1
1

K iB P
V2
Q

14

15

16

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Deriving the rate equations for product inhibition

Rapid equilibrium random Bi Bi with dead-end EBQ
complex


1
1
KB

1
A:
vo V 1
B




KA
K iB
Q
Q 1 17
1
1

K iQ
K iiQ A
V1
B

B:




1
1
KA
Q
1

1
vo V 1
K iiQ
A



KB
K iA
Q
1
1
1

K iQ B
V1
A

C:

D:

E:

F:

G:

H:



1
1
KA

1
vo V 1
A



KB
K iA
P
1
1

1
K iP B
V1
A


1
1
KP

1
vo V 2
P



KQ
K iP
A
1
1

1
K iA Q
V2
P


1
1
KQ

1
vo V 2
Q



KP
K iQ
A
1
1
1

K iA P
V2
Q



1
1
KP
B
1

1
vo V 2
K iiB
P



KQ
K iP
B
1
1

1
K iB Q
V2
P

Rapid equilibrium random BiBi with dead-end EAP and

EBQ complexes


1
1
KB

1
A:
vo V 1
B




KA
K iB
Q
Q 1
1
1

25
K iQ
K iiQ A
V1
B

B:




1
1
KA
Q
1

1
vo V 1
K iiQ
A



KB
K iA
Q
1
1
1

K iQ B
V1
A

18

26

19




1
1
KB
P
1

1
vo V 1
K iiP
B



KA
K iB
P
1
1

1
K iP A
V1
B

20



1
1
KA

1
vo V 1
A




KB
K iA
P
P 1 28
1

K iP
K iiP B
V1
A

21



1
1
KP

1
vo V 2
P




KQ
K iP
A
A 1 29
1

K iA
K iiA Q
V2
P

1
1
KB

1
vo V 1
B



KA
K iB
P
1
1
1

K iP A
V1
B

621

C:

D:

E:

F:
22

G:
23



1
1
KQ

1
vo V 2
Q




KP
K iQ
B
B 1
1

24
K iB
K iiB P
V2
Q

H:




1
1
KQ
A
1

1
vo V 2
K iiA
Q



KP
K iQ
A
1
1

1
K iA P
V2
Q



1
1
KP
B
1

1
vo V 2
K iiB
P



KQ
K iP
B
1
1

1
K iB Q
V2
P

27

30

31



1
1
KQ

1
vo V 2
Q




KP
K iQ
B
B 1 32
1

K iB
K iiB P
V2
Q

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622 V. Leskovac et al.

Rapid equilibrium random Bi Bi with dead-end EAP,
EBQ, PE, and PEP complexes


1
1
KB

1
A:
vo V 1
B




KA
K iB
Q
Q 1 33
1
1

K iQ
K iiQ A
V1
B

B:




1
1
KA
Q
1

1
vo V 1
K iiQ
A

D:




KQ
K iP
B
P2
1

K iB aK x K iP
V2
P

K iP 1

39
Kx Q

H:




1
1
KQ
K iP
1

1
vo V 2
Q
Kx

34




KP
K iQ
B
P2
1

K iB aK x K iP
V2
Q

B 1

40
K iiB P




K iB
P
P
P2
1
35
1
1

K iP K X aK X K iP A
B

Rapid equilibrium ordered Bi Bi without dead-end

complexes

C:




KB
K iA
Q
1
1
1
K iQ B
V1
A

G:




1
1
KP
B
1

1
vo V 2
K iiB
P




1
1
KB
P
KA
1

vo V 1
K iiP
B
V1



1
1
KA
KB

vo V 1
A
V1




K iA
P
P
P2
P 1
1
1

K iP K X aK X K iP
K iiP B
A
36



1
1
KP
E:

1
vo V 2
P




1
1
KQ
A
K iP
F:
1

vo V 2
K iiA K x
Q



KP
K iQ
A
P2
1
1
1

K iA aK x K iP P
V2
Q





1
1
KB
K iA K B
Q 1
41
1

vo V 1
K iQ A
B
V1 B

B:




1
1 KB
K iA
Q
1
1

1
vo V 1 V 1
K iQ B
A

42


 

1
1
KB
K iA K B 1
C:

vo V 1
B
V1 B A

43



1
1 KB
K iA 1

1
vo V 1 V 1
A B

44

D:




KQ
K iP
A
P
P2
1
1

K iA K x aK x K iP
V2
P

A 1

37
K iiA Q

A:

E:





1
1
KP
K iQ K P
A 1
45
1

vo V 2
K iA Q
P
V2 P




1
1 KP
K iQ
A
1
F:
1

1
vo V 2 V 2
K iA P
Q

46

G:


 

1
1
KP
K iQ K P 1

vo V 2
P
V2 P Q

47

H:



1
1 KP
K iQ 1

1
vo V 2 V 2
Q P

48

38

Deriving the rate equations for product inhibition

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Rapid equilibrium ordered Bi Bi with dead-end EAP

complex.




1
1
KB
K iA K B
Q 1
A:
1

vo V 1
K iQ A
B
V1 B

B:




1
1
KB
K iA
Q
1

1
1
vo V 1 V 1
K iQ B
A

49

50

Rapid equilibrium ordered Bi Bi with dead-end EBQ

complex


1
1
KB
K iA K B

A:
vo V 1
B
V1 B



Q
B
1
1
1
57
K iQ
K iiB A

B:



1
1
K iA K B Q

1
vo V 1
K iiB K iQ A



KB
K iA
Q
1
1
1
K iQ B
V1
A

58

C:


 

1
1
KB
K iA K B 1

vo V 1
B
V1 B A

59

D:



1
1
KB
K iA 1

1
vo V 1 V 1
A B

60

C:



 

1
1
KB
P
K iA K B 1
51
1

vo V 1
K iiP
B
V1 B A



1
1 KB
K iA
P 1
D:

1
vo V 1 V 1
A K iiP B

E:



1
1
KP
K iQ K P

vo V 2
P
V2 P



A
P
1
1
1
K iA
K iiP Q



1
1
K iQ K P A
KP
F:

vo V 2
K iA K iiP Q
V2



K iQ
A
1
1
1
K iA P
Q

52

E:
53

F:


 

1
1
KP
K iQ K P 1
G:

vo V 2
P
V2 P Q



1
1 KP
K iQ 1

1
vo V 2 V 2
Q P



1
1
KP

1
vo V 2
P


K iQ K P
A 1
1

K iA Q
V2 P

61




1
1
KP
K iQ
A
1
1

1
vo V 2 V 2
K iA P
Q

62




1
1
KP
B
1

1
vo V 2
K iiB
P


K iQ K P 1

V2 P Q

63



1
1
KP
K iQ
B
1

1
vo V 2 V 2
K iiB P
Q

64

54

G:

H:

623

55

56

H:

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624 V. Leskovac et al.

Forward rapid equilibrium random reverse rapid
equilibrium ordered Bi Bi


1
1
KB

1
A:
vo V 1
B



KA
K iB
Q
1
1
1

65
K iQ A
V1
B

B:



1
1
KA

1
vo V 1
A



KB
K iA
Q
1
1

1
K iQ B
V1
A

Forward rapid equilibrium ordered reverse rapid

equilibrium random Bi Bi




1
1
KB
K iA K B
Q 1
73

1
A:
vo V 1
K iQ A
B
V1 B




1
1 KB
K iA
Q
1
B:
1

1
vo V 1 V 1
K iQ B
A

66
C:

C:

D:





1
1
KB
KA
K iB 1
67

1
1

vo V 1
B
V1
B A





1
1
KA
KB
K iA 1
68

1
1

vo V 1
A
V1
A B





1
1
KP
K iQ K P
A 1
E:
69
1

vo V 2
K iA Q
P
V2 P




1
1 KP
K iQ
A
1
F:
1

1
vo V 2 V 2
K iA P
Q





1
1
KB
K iA K B
P 1
75
1

vo V 1
K iP A
B
V 1B




1
1 KB
K iA
P
1
D:
1

1
vo V 1 V 1
K iP B
A



1
1
KP
E:

1
vo V 2
P



KQ
K iP
A
1
1

1
K iA Q
V2
P

F:
70

74



1
1
KQ

1
vo V 2
Q



KP
K iQ
A
1
1

1
K iA P
V2
Q

76

77

78





1
1
KP
K iQ K P
B 1
G:
71
1

vo V 2
K iB Q
P
V2 P





1
1
KP
KQ
K iP 1
G:

1
1

vo V 2
P
V2
P Q

79




1
1 KP
K iQ
B
1
H:
1

1
vo V 2 V 2
K iB P
Q

H:





1
1
KQ
KP
K iQ 1

1
1

vo V 2
Q
V2
Q P

80

72

625

Deriving the rate equations for product inhibition

Rapid equilibrium ordered Bi Bi with dead-end QE and
QEQ complex
F:


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A:

1
1
KB

1
vo V 1
B

K iA K B
V1 B



Q
Q
Q2
1

1
K iQ K X aK X K iQ A




1
1
KP
K iQ
A
1

1
vo V 2 V 2
K iA
Q

K iQ
Q 1

K X aK X P

81



1
1
KP
K iQ
1

1
vo V 2
P
KX

G:
B:

1
1 KB

vo V 1 V 1



K iA
Q
Q
Q2
1
82
1
1

K iQ K X aK X K iQ B
A


 

1
1
KB
K iA K B 1
C:

vo V 1
B
V1 B A

83



1
1 KB
K iA 1
D:

1
vo V 1 V 1
A B

84

E:




1
1
KP
K iQ
1

1
vo V 2
P
KX



K iQ K P
A
Q2
1
1

K iA aK X K iQ Q
V 2P

86

85



K iQ K P
Q2
1
1

V 2P
aK X K iQ Q

87



1
1 KP
K iQ K iQ
Q
1

H:
vo V 2 V 2
KX
Q
aK X P

88

Product inhibition patterns. From Equations (1 88),

one can obtain 88 different product inhibition
patterns, 44 in the forward and 44 in the reverse
direction. Thus, the product inhibition patterns listed
in Table II, are constructed from the product inhibition
Equations (1 88). The primary plots, listed in Table II,
show the effect of product inhibitors on intercepts in
double-reciprocal plots. Note that for mechanisms
1 5, the patterns in the forward direction are
symmetrical to patterns in the reverse direction; in
mechanisms 6 11 this symmetry is absent. All double
reciprocal plots (primary plots) are linear.
Table III shows the effect of inhibitors on slope
replots; slope replots are obtained from the primary

Table II. Effect of product inhibitors on intercepts of primary plots in rapid equilibrium Bi Bi mechanisms.
Product inhibitor
Q

Product inhibitor

Varied substrate

Varied substrate

Mechanism

1/A

1/B

1/A

1/B

1/Q

1/P

1/Q

1/P

Experiment

1
2
3
4
5
6
7
8
9
10
11

C
C
C
C
C
C
C
C
C
C
C

C
C
NC
NC
NC
C
C
NC
C
C
C

C
NC
C
NC
NC

UC

C
C
C
C
C

C
C
C
C
C
C
C
C
C
C
C

C
NC
C
NC
NC
C
NC
C
C
C
C

C
C
NC
NC
NC

UC
C

C
C
C
C
C

C
C

RE Random Bi Bi without dead-end complexes

RE Random Bi Bi with EAP complex
RE Random Bi Bi with EBQ complex
RE Random Bi Bi with EAP and EBQ complexes
RE Random Bi Bi with EAP, EBQ, PE, and PEP complexes
RE Ordered Bi Bi without dead-end complexes
RE Ordered Bi Bi with dead-end EAP complex
RE Ordered Bi Bi with dead-end EBQ complex
Forward RE random, reverse RE ordered Bi Bi
Forward RE ordered, reverse RE random Bi Bi
RE ordered Bi Bi, with QE and QEQ complex

RE rapid equilbirium; C competitive; NC noncompetitive; UC uncompetitive; product inhibitor has no effect on intercept
or slope.

626 V. Leskovac et al.

Table III. Slope replots in product inhibition patterns in rapid equilibrium Bi Bi mechanisms.
Product inhibitor
Q

Product inhibitor

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Varied substrate

B
Varied substrate

Mechanism

1/A

1/B

1/A

1/B

1/Q

1/P

1/Q

1/P

Experiment

L
L
L
L
L
L
L
L
L
L
NL

L
L
L
L
L
L
L
L
L
L
NL

L
L
L
L
NL
L/N
L/N
L/N
L/N
L
L/N

L
L
L
L
NL
L/N
L
L/N
L/N
L
L/N

L
L
L
L
L
L
L
L
L
L
L

L
L
L
L
L
L
L
L
L
L
L

L
L
L
L
L
L/N
L/N
L/N
L
L/N
L/N

L
L
L
L
L
L/N
L/N
L
L
L/N
L/N

1
2
3
4
5
6
7
8
9
10
11

RE Random Bi Bi without dead-end complexes

RE Random Bi Bi with EAP complex
RE Random Bi Bi with EBQ complex
RE Random Bi Bi with EAP and EBQ complexes
RE Random Bi Bi with EAP, EBQ,PE, and PEP complexes
RE Ordered Bi Bi without dead-end complexes
RE Ordered Bi Bi with dead-end EAP complex
RE Ordered Bi Bi with dead-end EBQ complex
RE Forward random, RE reverse ordered Bi Bi
RE Forward ordered, RE reverse random Bi Bi
RE ordered Bi Bi, with QE and QEQ complex

SS steady-state; RE rapid equilibrium; L linear; NL nonlinear; L/N slopes in primary plots are linear, but not responding to
increasing concentrations of product inhibitor.

Steady-state Bi Bi mechanisms

The derivation of rate equations for steady-state

mechanisms is a difficult task compared to rapid
equilibrium mechanisms. For bisubstrate mechanisms with two substrates and two products of
reaction, it is necessary to use the manual procedures,
the King-Altman method [12] the method of [1], or
other methods [14]. The derivations are lengthy and
require some pratice for an efficient application. Let
us review the rate equations for steady-state
mechanisms.

In this section, we shall consider seven steady-state or

mixed rapid equilibrium and steady-state mechanisms: steady-state ordered, Theorell-Chance, steady-

Mechanism 12. Steady-state ordered (Scheme 4).

plots. The nonlinear slope replots in secondary plots

are obtained only in two cases, when one of reactants
binds twice to the enzyme, in a sequential fashion
(Mechanisms 4 and 11 in Table I). Thus, combining the
informations obtained from primary and secondary
plots, one may unambiguously identify each mechanism
in Table I and distinguish it clearly from all others.

Scheme 4.

state ordered with a rapid equilibrium segment,

steady-state ordered with dead-end EB complex,
steady-state ordered with dead-end EP complex,
forward rapid equilibrium random and reverse
steady-state ordered, and the steady-state random.

In this mechanism, the binding of substrates is

ordered from both sides of reaction. The full rate
equation is complex and contains more denominator
terms then enzyme forms in the mechanism; contrary to
that, the rapid equilibrium mechanisms always contain

Deriving the rate equations for product inhibition

one denominator term for each enzyme form in the
mechanism.

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Mechanism

13.
A

Theorell-Chance
B

EA

(Scheme

5).

EQ

Scheme 5.

The Theorell-Chance mechanism is a simplified

version of an ordered mechanism where the steadystate level of central complexes is very low. Therefore,
the rate equatioin is identical with the ordered
mechanism, except that the terms in ABP and BPQ
are missing from the denominator.

Mechanism 14. Steady-state ordered with rapid

equilibrium segments (Scheme 6).

627

In Scheme 6 the shaded area represents the rapid

equilibrium segments. The rate equation (Equation V)
has the same form as that for the rapid equilibrium
ordered mechanism (Mechanism 6 in Table I), except
that the kinetic constants associated with B and P are
the Michaelis constants and the constants associated
with A and Q are true dissociation constants.

Mechanism 15. Steady-state ordered with dead-end EB

complex. In this ordered mechanism B binds to E to
form an unproductive complex EB which cannot bind
A. The rate equation has the same form as that for the
steady-state ordered Bi Bi mechanism, except that
denominator terms associated with V2KiAKB, B and P
are multiplied with (1 B/KX). A new constant KX is
the dissociation constant of B from the EB complex.

Mechanism 16. Steady-state ordered with dead-end EP

complex. In this ordered mechanism P binds to E to
form an unproductive complex EP which cannot bind
Q. The rate equation again has the same form as that
for the steady-state ordered Bi Bi mechanism, except
that denominator terms associated with V2KiAKB, B
and P are multiplied with (1 P/KX). KX is the
dissociation constant of P from the EP complex.

Mechanism 17. Forward rapid equilibrium random,

reverse steady-state ordered (Scheme 7). This
mechanism was described by [10] in order to explain
the kinetic mechanism of Asp49 mutant of yeast
alcohol dehydrogenase.

Scheme 6.

Mechanism 18. Steady-state random Bi Bi mechanism.

The last mechanism, a steady-state random Bi Bi,
makes an exception to all other mechanisms. A full

Scheme 7.

628 V. Leskovac et al.

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rate equation for this mechanism contains four dozen

of kinetic terms in the denominator and, therefore, it
is completely unsuitable for practical work [14,17]
consequently, the full rate equation is not presented.

F:

Derivation of rate equations in the double-reciprocal form

The derivation of rate equations in the doublereciprocal form from the full rate equations is
essentially the same as for the rapid equilibrium
reactions. First, the terms in the denominator, which
contain the product that is omitted, are deleted. Then,
the remaining equation is inverted and sorted out into
8 product inhibition equations. Since we have 7 full
rate equations (Mechanism 18 is not shown), a total of
7 8 56 product inhibition equations are presented below (Equations 89 136).

Steady-state ordered


1
1
KB
A:

1
vo V 1
B



KA
K iA K B
Q 1

1
1
K iQ A
V1
K AB

B:

C:

H:

89




1
1
KA
Q
1

1
vo V 1
K iQ
A



KB
K iA
Q
1
1
1

K iQ B
V1
A

G:

E:

B:

96

97

92

D:

93




1
1
KA
Q
1

1
vo V 1
K iQ
A



KB
K iA
Q
1
1
1

K iQ B
V1
A

C:





1
1
KP
KQ
K iQ K P

1
1

vo V 2
P
V2
K QP


A 1
1
K iA Q



1
1
B
KQ

vo V 2
K iB
Q



KP
K iQ
K AB 1
1

1
K iA K B P
V2
Q

90





1
1
KB
K QP
P
1

vo V 1
K iQ K P
K iP
B



1
1
KA
P

1
vo V 1
K iP
A



KB
K iA
K QP 1

1
1
K iQ K P B
V1
A

94




1
1
B
KP
K AB
1

vo V 2
K iB
K iA K B
P



KQ
K iQ K P
K AB
1 95

1
1
K iA K B Q
V2
K QP

Theorell-Chance


1
1
KB

1
A:
vo V 1
B



KA
K iB
Q 1
1

1
K iQ A
V1
B




KA
K iA K B
K QP
1 91
1
1

K iQ K P A
V1
KA B

D:




1
1
KQ
A
1

1
vo V 2
K iA
Q



KP
K iQ
A
1

1
1
K iA P
V2
Q




1
1
KB
P
1

1
vo V 1
K iP
B



KA
K iB
P
1
1

1
K iP A
V1
B





1
1
KA
KB
K iA

1
1

vo V 1
A
V1
A


P 1

1
K iP B

98

99

100

Deriving the rate equations for product inhibition

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E:

F:

G:

H:



1
1
KP

1
vo V 2
P



KQ
K iP
A 1
1

1
K iA Q
V2
P



1
1
KQ
A
1

1
vo V 2
K iA
Q



KP
K iQ
A
1
1

1
K iA P
V2
Q



1
1
KP
B
1

1
vo V 2
K iB
P



KQ
K iP
B
1
1

1
K iB Q
V2
P


1
1
KQ

1
vo V 2
Q



KP
K iQ
B 1

1
1
K iB P
V2
Q

101

102

103

A:

B:

Steady-state ordered with a dead-end EB complex.



1
1
KB

1
A:
vo V 2
B



KA
K iA K B
Q
B 1

1
113
K iQ K X A
V1
K AB



1
1
KA
Q K iA K B
B:
1

vo V 1
K iQ K A K X
A




KB
K iA
Q
K AB 2 1
114
1
1

K iQ
V1
A
K BK XA B

C:



1
1 KB

vo V 1 V 1



K iA
Q
1
1
1
K iQ B
A

B
KX




K iA K B
K QP
1
1
1
K iQ K P
A
K AB

104

115

D:





1
1
KB
K iA K B
Q 1
105
1

vo V 1
K iQ A
B
V 1B





1
1
P
KB
K QP
KA
1

vo V 1
K iP B
K iQ K P
V1

Steady-state ordered with a rapid equilibrium segment

629


1
1
P
KA

vo V 1
K iP A



K iA K B
K QP
1
1
K iQ K P
K AK X

106

KB
V1





K QP
K iA
K AB 2 1
1
1

K iQ K P
A
K BK XA B
116

C:


 

1
1
KB
K iA K B 1

vo V 1
B
V 1B A

107

D:



1
1 KB
K iA 1

1
vo V 1 V 1
A B

108

E:

1
1
KP

1
vo V 2
P


F:



K iQ K P
A 1
109
1

K iA Q
V 2P


1
1 KP
K iQ
A
1

1
vo V 2 V 2
K iA
Q




 

1
1
KP
K iQ K P 1
G:

vo V 2
P
V 2P Q


1
1 KP
K iQ 1
H:

1
vo V 2 V 2
Q P

1
P

E:

F:



1
1
KP

1
vo V 2
P



KQ
K iQ K P
A 1
1

1
K iA Q
V2
K QP



1
1
KQ
A
1

1
vo V 2
K iA
Q



KP
K iQ
A
1
1

1
K iA P
V2
Q

117

118

110

111

112





1
1
KP
K AB
B
G:
1

vo V 2
K iA K B
K iB
P



KQ
K iQ K P
K AB
1

1
K iA K B
V2
KQ P


B
1
KX



1
Q

119

630 V. Leskovac et al.

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H:





1
1
KQ
B
B
1

vo V 2
KX
K iB
Q



KP
K AB
K iQ
K AB
1

K iA K B
K iA K B
V2
Q


B
1
1
120
KX P

Steady-state ordered Bi Bi with a dead-end Ep complex





1
1
KB
KA
K iA K B
A:

1
1

vo V 1
B
V1
KA B


Q 1
121
1
KiQ A



1
1
KA
Q
1
B:

1
vo V 1
K iQ
A

C:




KB
K iA
Q
1
1
1
K iQ B
V1
A

122





1
1
KB
K QP
P
1

vo V 1
K iQ K P
K iP
B
KA

V1




K iA K B
K QP
1
1
K iQ K P
KA B



P
1
1
KX A
D:

E:

1
1
KP

1
vo V 2
P

KQ
K iQ K P
1
V2
K QP





1 1
B KQ
K iQ K p
K AB
1

1
vo V 2
K iB Q
K iA K B
K QK X

KP
V2





1
1
KB
K QP
P
KA
1

vo V 1
K iQ K P
K iP
B
V1




K iB
K QP
B
1
1
1
1
131
K iQ K P
K iB
A
B

D:





1
1
KA
K QP
P
1

vo V 1
K iQ K P
K iP
A

125




1
1
KQ
A
K iQ K P
1

vo V 2
K iA K Q K X
Q




KP
K iQ
A
K QP 2 1
1
1

K iA
V2
Q
K PK XQ P
126





K AB
K iQ
K QP 2 1
1
1

K iA K B
Q
K PK XQ P
128

Forward rapid equilibrium random, reverse steady-state

ordered


1
1
KB
A:

1
vo V 1
B



KA
K iB
Q
1
1

1
129
K iQ A
V1
B


1
1
KA
B:

1
vo V 1
A



KB
K iA
Q
1
130
1
1

K iQ B
V1
A

124



A
P 1

1
K iA K X Q

F:

H:

C:



P
1
1
KX B


127

123





1
1
KA
P
P
1

vo V 1
KX
K iP
A



KB
K QP
K iA
K QP
1

K iQ K P
K iQ K p
V1
A




1
1
B
KP
K AB
KQ
1

G:
vo V 2
K iB P
K iA K B
V2




P
K iQ K P
K AB
1
1
1
1
KX
K iA K B
Q
K QP




KB
K QP
K iA 1
1
1
K iQ K P
V1
A B



1
1
KP
KQ
E:

vo V 2
P
V2



A K iQ K P
A
1
1
1

K iA
K iA Q
K QP
F:




1
1
KQ
A
1

1
vo V 2
K iA
Q



KP
K iQ
A
1
1

1
K iA P
V2
Q

132

133

134

631

Deriving the rate equations for product inhibition

Table IV. Effect of product inhibitors on intercepts of primary plots in steady-state Bi Bi mechanisms.
Product inhibitor
Q

Product inhibitor
P

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Varied substrate

Varied substrate

Mechanism

1/A

1/B

1/A

1/B

1/Q

1/P

1/Q

1/P

Experiment

C
C
C
C
C
C
NC

NC
NC
C
NC
NC
C
NC

NC
NC

NC
NC
NC
NC

NC
C

NC
NC
NC
NC

C
C
C
C
C
C
NC

NC
NC
C
NC
NC
NC
NC

NC
NC

NC
NC
C
NC

NC
C

NC
NC
NC
NC

12
13
14
15
16
17
18

SS Ordered Bi Bi
SS Theorell-Chance
SS Ordered Bi Bi with RE segment
SS Ordered Bi Bi with dead-end EB complex
SS Ordered Bi Bi with dead-end EP complex
Forward RE random, reverse SS ordered Bi Bi
SS Random Bi Bia)

SS steady-state; RE rapid equilbirium; C competitive; NC noncompetitive; product inhibitor has no effect on intercept or
slope.
a)
All patterns are noncompetitive [Segel, 1975].

of product inhibitors on primary plots (intercept

effects), and Table V shows the effect of inhibitors on
secondary plots (slope effects).
Finally, we can conclude that, combining the
information obtained from primary and secondary
plots in Tables II V, one may unambiguously identify
each of the 18 bisubstrate mechanisms and distinguish
it clearly from all others.



1
1
KP
G:

1
vo V 2
P



KQ
B K iQ K P
B
1 135
1
1

K iB
K iB Q
V2
K QP

H:




1
1
KQ
B
1

1
vo V 2
K iB
Q



KP
K iQ
B
1
1

1
K iB P
V2
Q

Practical examples
136

In this section, we shall illustrate the practical

application of product inhibition studies, outlined
above, for solving the kinetic mechanism of a specific
enzyme. In doing so, we have chosen different
preparations of yeast alcohol dehydrogenase from
bakers yeast operating with different substrates. For
the purpose of this study, three examples were

Product inhibition patterns. The construction of

product inhibition patterns from product inhibition
equations is the same as in the case of rapid
equilibrium mechanisms. Table IV shows the effect

Table V. Slope replots in product inhibition patterns in steady-state Bi Bi mechanisms.

Product inhibitor
Q

Product inhibitor
P

Varied substrate

B
Varied substrate

Mechanism

1/A

1/B

1/A

1/B

1/Q

1/P

1/Q

1/P

Experiment

L
L
L
L
L
L
NL

L
L
L
L
L
L
NL

L
L
L/N
L
NL
L
NL

L
L
L/N
L
NL
L
NL

L
L
L
L
L
L
NL

L
L
L
L
L
L
NL

L
L
L/N
NL
L
L
NL

L
L
NL
NL
L
L
NL

12
13
14
15
16
17
18

SS Ordered Bi Bi
SS Theorell-Chance
SS Ordered Bi Bi with RE segment
SS Ordered Bi Bi with dead-end EB complex
SS Ordered Bi Bi with dead-end EP complex
Forward RE random, reverse SS ordered Bi Bi
SS Random Bi Bia)

SS steady-state; RE rapid equilbirium; L linear; NL nonlinear; L/N slopes in primary plots are linear, but not responding to
increasing concentrations of product inhibitor.
a)
All patterns are nonlinear, but the non-linearity is difficult to detect.

632 V. Leskovac et al.

Table VI. Product inhibition patterns in primary plots in practical examples.
Substrate

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Experiment
A
B
C
D
E
F
G
H

Variable

Fixed

Product
inhibitor

A
B
A
B
Q
P
Q
P

B
A
B
A
P
Q
P
Q

Q
Q
P
P
A
A
B
B

Example 1

Example 2

Example 3

Wild type
Ethanol/acetaldehydea) [9]

Asn49 mutant
Ethanol/acetaldehydea) [10]

Wild type
propan-2-ol/
acetoneb) [18]

C
C
NC

NC
C

NC

C
NC
NC

C
NC
NC
C
C
NC
NC
C

C competitive; NC noncompetitive.
a)
At pH 7.3;b) At pH 7.0.

examined which are dealing extensively and in detail

with kinetic studies of yeast alcohol dehydrogenase
reactions (Table VI).
Table VI shows the product inhibition patterns for
three examples of the yeast enzyme-catalyzed reactions from the literature: the oxidation of ethanol by
NAD and the wild type yeast alcohol dehydrogenase
([9]), the oxidation of ethanol by NAD and the
Asn49 mutant of the same enzyme [10], and the
oxidation of propan-2-ol by NAD and the wild type
yeast alcohol dehydrogenase [18].
The assignment of substrates in Table VI was the
same as outlined in the above sections: A was assigned
to oxidized coenzyme and B to alcohol substrate in the
forward direction, P was assigned to carbonyl substrate
and Q to reduced coenzyme in the reverse direction.

Example 1
Oxidation of ethanol by NAD and the wild type yeast
alcohol dehydrogenase [9]. The product inhibition
patterns in primary plots in this case are compatible
with the steady-state ordered Bi Bi mechanism
(Mechanism 12); acetaldehyde could be shown to be
noncompetitive against ethanol only in very precise
experiments [19]. The kinetic isotope effects in the
forward direction were DV1 1.8, DV1/KA 1.8, and
D
V1/KB 3.2, compatible with the ordered addition of
substrates [10]. In the reverse direction, the kinetic
isotope effects were close to unity, DV2 0.7, DV2/KQ
0.9, and DV2/KP 1.3, suggesting the absence of
isotope-sensitive steps in this direction [10]. The still
significant value of DV1/KA is probably due to
dissociation of NAD from the ternary complex, as
suggested by [7].
A further kinetic complication is a double-reciprocal plot for inhibition of acetaldehyde by ethanol,
which is noncompetitive, but the lines have no
common intersection point to the left of the vertical
axis as was shown by [8]; this indicates the presence of

an extra complex in the mechanism. In summary, the

kinetic isotope effects are compatible with the steadystate ordered mechanism overall, with some dissociation of reduced coenzyme from the ternary
complex.

Example 2
Oxidation of ethanol by NAD and Asn49 mutant of
yeast alcohol dehydrogenase ([10]). The product
inhibition patterns in primary plots in this case are
compatible with the forward rapid equilibrium
random and reverse steady-state ordered Bi Bi
mechanism (Mechanism 17).
One kinetic complication, reported by [10], is the
observation of a noncompetitive double-reciprocal
plot for inhibition of NADH by ethanol (entry G in
Table VI), which however, should be competitive
according to Equation (135). The primary kinetic
isotope effects in the forward direction, DV1 2.1,
D
V1/KA 2.2, and DV1/KB 2.3 were compatible
with the random addition of substrates. In the reverse
direction the kinetic isotope effects were not significantly different from the wild type enzyme, DV2 1.2,
D
V2/KQ 1.3, and DV2/KP 1.4, suggesting high
commitment factors in the reverse direction; these
results are not consistent with the rapid equilibrium
assumption. In summary, it appears that Mechanism
17 describes the simplest case compatible with
experimental data.

Example 3
Oxidation of propan-2-ol by NAD and wild type
yeast alcohol dehydrogenase [15,16,18]. The product inhibition patterns in primary plots, for this
case, are in agreement with mechanisms 4, 5 and 13
(Table I, Scheme 5). Since the slope replot for
experiment D in Table IV was nonlinear (Figure 1),
it was concluded that Mechanism 5 in Table I, a

Deriving the rate equations for product inhibition

633

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Conclusions

Figure 1. Fit of the slope function from Equation (VII) ()

to experimentally determined slope function (o) for inhibition of
propan-2-ol with acetone, at constant NAD, which were measured
in experiment [18].

rapid equilibrium random mechanism with two

additional dead-end complexes, PE and PEP, was
in question.
A further kinetic complication in this case was the
observation of a linear slope replot in experiment C in
Table VI (data not shown), which however, should be
nonlinear according to the Mechanism 5 in Table IV;
this corresponds to Equation (35). Therefore, the
Mechanism 5 in Table I must be regarded only as the
closest description of experimental data.
Further kinetic analysis was performed by fitting the
slope function in Equation (VI) for variable B
(propan-2-ol) in the presence of constant A (NAD)
and increasing concentrations of P (acetone), to the
slope data from the experiment.


1
1
KA
KB

vo V 1
A
V1




K iA
P
P
P2
1
1

K iP K X aK X K iP
A

P 1

K iiP B

VII

Figure 1 shows the fit of Equation (VII) to

experimental data; Equation (VII) is identical to
Equation (36). The fixed kinetic constants in this
equation, V1 7 s -1, K iA 0.38 mM, KiB
117 mM, KP 477 mM, KiP 194 mM, and
KiiP 170 mM, were calculated from the data of [18]
and inserted back into the equation. After this, the
constants KX and a were obtained from the best fit.
From the Figure 1 one can see that the combination
of KX 13.5 mM and a 1, gives the best fit.
However, other pairs of constants (such as
KX 10 mM;a 2) provide a good fit as well which
indicates that the two constants cannot be determined
independently.

Product inhibition experiments are often the best means

of distinguishing different mechanisms. However, they
have their limitations which are illustrated above, in the
practical section, with examples from the yeast alcohol
dehydrogenase kinetics. The main limitations of the
product inhibition diagnostics are as follows.
First, it is usually very difficult to detect the nonlinearity of the primary plots if it is present. Second, it is
easy to overlook the multiple intersection points
in primary plots left to the vertical axis, if they are
present. Third, it is sometimes difficult to detect a
difference between the competitive and noncompetitive product inhibition pattern in primary plots,
especially if the Michaelis and inhibition constant for
the substrate are widely different. Fourth, it may be
sometimes difficult to detect the non-linearity in slope
or intercept replots in secondary plots. A failure to
observe any of these anomalies will lead to erroneous
conclusions about the mechanism involved.
There are two main sources of error in arriving at a
correct mechanism from experimental data. First, one
may easily overlook the existence of one (or more)
enzyme-substrate complexes which were omitted from
the mechanism. On the other hand, all enzyme forms
may be accounted for in the mechanism, but the rapid
equilibrium mechanism may have a steady-state
segment and vice versa, a steady-state mechanism
may have a rapid equilibrium segment. In both cases,
the full rate equation for a real mechanism may
become more complex and sometimes unmanageable
for practical purposes.
For this reason, the analysis of a given kinetic
mechanism, obtained with product inhibition studies,
must be always expanded with other methods,
preferably by the use of dead-end inhibitors and
especially by the application of kinetic isotope effects
[5,14].
The product inhibition patterns shown in
Tables II V have appeared to the extend of approximately one third in textbook format in Segel (1974)
and in [14] books. The product inhibition equations
(Equations 1 136) have appeared by approximately
one fifth in the above textbooks. Therefore, this article
is by far the most complete survey of product inhibition
patterns and equations published so far.
Acknowledgements
This work was financially supported by the Ministry
of Science and Environmental Protection of the
Republic of Serbia, Project No 142046.

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