BIOC 301 - Lecture 3 - Olson - More Complex Kinetic Patterns

(LP0B-6, pp. 206-207; mostly these notes; see other Biochemistry Textbooks)

A. Bisubstrate reactions (the most common processes)

1. General equations (can be derived from principles in Olson-Lecture 1, p. 8-10):
A + B
P + Q
There are a large variety of ways of adding and removing both reactants and products. The enzyme can
act simply as a template for both reactants or actively participate as an intermediate in the reaction. The
general steady state equation is:

Vmax[A][B]
, where the K's are referred to as "Michaelis" constants and are often
Kab + Ka [B] + Kb [A] + [A][B]
complex functions of individual rate constants for specific steps in the reaction mechanism. Rearranging:
vi =

Vmax [B]
[A]
K b + [B]
vi =
K ab + K a [B]
+ [A]
K b + [B]

vi =
obs
Vmax
=

Analysis: Fix B and vary A and then

repeat the experiment at another [B] to
see how KM(obs) and Vmax(obs) depend on
the concentration of the second substrate.

obs
Vmax
[A]

K obs
M + [A]

Vmax [B]
K ab + K a [B]
; K obs
M =
K b + [B]
K b + [B]

Remember the reciprocal form is:

K obs
1
1
1
M
= obs + obs
vi
[A]
Vmax Vmax

2. Two classes of bisubstrate reactions: Ternary and Binary Complex Mechanisms (significant
theoretical and experimental differences)
a.) Ternary complex mechanisms (other names: sequential bi bi (Cleland nomenclature for two
consecutive bimolecular steps) or single displacement reactions)
i.) General scheme for a rapid equilibrium scheme:
For this mechanism, the enzyme is acting like a
template rather than a donor or acceptor.
A
KA
B

E
KB

EA

K'B
A
EB

kcat
EPQ

EAB

Release of products P and Q

K'A

Kab

[E][A]
[E][B]
[E][A][B]
; KB =
; K AB =
[EA]
[EB]
[EAB]
The ternary complex is required for product formation from both substrates.
d[P]

v i =
= kcat [EAB]. There is no covalent enzyme-partial substrate intermediate.
dt t = 0
KA =

ii.) The kinetic characteristics of ternary complex mechanisms are converging lines in
double reciprocal plots. This means that Kab has a finite value and the species EAB exists.
Experimentally, [B] affects the slope of a 1/vi vs. 1/[A] plot.
B1

Vmax[B]
[A]
Kb + [B]
and
vi =
Kab + Ka [B]
+ [A]
K b + [B]
1
1
= obs +
vi
Vmax

K obs
M
obs
Vmax

1/vi
B2

1
,
[A]

Converging
lines
B3
increasing
[B]

where Vmax(obs) and KM(obs) show different

dependencies on [B].
This test holds whether or not the rapid
equilibrium assumption is valid.
1/[A]

1/Vmax (obs)

- 1/KM (obs)
B. Two subsets or extremes of the ternary complex mechanism.

1. Random sequential addition of substrates - the binding of B is uninfluenced by bound A and

vice versa. The enzyme is simply acting as a template. For the rapid equilibrium assumption,
Kab = KA M
#B. (Problem Set 3, question 3 like noncompetitive inhibition)
a) Examples: most kinases, including creatine kinase
H2
C
O

C
O
(-)

NH
N

H2
C

CH3

O
NH2

ATP
B

(-) O

Creatine
A

NH
N
CH3

C
N
H

O
(-)

P
O
O
(-)

ADP
Q

Creatine-Pi
P

b) Derivation assuming rapid equilibria for substrate binding and the scheme on page 1. (See
Appendix C, Problem Set 3, question 3.d)

v i = k cat [EAB]
[E][A]
[EB][A]
= KA =
KA =
[EA]
[EAB]
[E][B]
[EA][B]
KB =
= KB =
[EB]
[EAB]
[E]o = [E] + [EA] + [EB] + [EAB]

Vmax [B]
Vmax [B]
[A]
[A]
K B + [B]
K B + [B]
=
vi =
K AK B + K A [B]
K A (K B + [B])
+ [A]
+ [A]
K B + [B]
K B + [B]
or
Vmax [B]
[A]
K B + [B]
; note K ab = K AK B
vi =
K A + [A]

c) Experimental test for a random ternary complex mechanism - lines intersecting on the xaxis in double reciprocal plots. The Kab term is equal to KAKB, and, as a result, KM(obs) does
not depend on [B], but Vmax(obs) does increase by a simple hyperbolic expression in [B].
Ternary Random

[B]1

increasing [B]
[B]2

Vmax[B]
[A]
K B + [B]
vi =
K A + [A] and
Remember :
K obs
1
1
1
M
= obs + obs
vi
Vmax Vmax [A]

[B]3

1/vi

[B]

KM(obs) shows no dependence on [B] and

the lines intersect on the x-axis.
Vmax(obs) does increase and, because
KM(obs) is unchanged, the slope decreases
with increasing [B]. Bottom line - B is
required for catalysis but does not affect the
binding of A.

K M (obs)
unchanging
Vmax(obs)increasing

1/[A]

d) Product inhibition pattern for a random ternary complex mechanism using creatine kinase as
an example. (Note, only one product can be added; otherwise the reaction would go backward).
Inhibitor fixed s

creatine

[A]

+ ATP

creatine-Pi +

[B]

[P]

Q
P
P
Q

ADP

[Q]

The non-competitive inhibition pattern suggests that

the binding of A and B is not ordered but random.

B
B
A
A

plot

pattern

1/vi vs. 1/[A]

1/vi vs. 1/[A]
1/vi vs. 1/[B]
1/vi vs. 1/[B]

noncompetitive
competitive
competitive*
competitive

*Since the Pi of creatine-Pi overlaps the -Pi of

ATP, the observed inhibition pattern will be
competitive (i.e., they both can't bind at the same
time).

2. An ordered sequential addition of substrates: B can't bind until A is bound and induces the
formation of the B binding site. The mechanism reduces to:
kcat
E + A

EA + B
KA

EAB

EPQ

release of products

K'B

a). Examples: most dehydrogenases, including lactate dehydrogenase. (Lecture 2, p. 8; Lecture 6, pp. 6, 7;
Problem Set 3, Question 4 like uncompetitive inhibition)

4
H2 N

H2 N
N

N
CH2

N
O

H H

O
O
O

CO2
+

O
NH2

P
O CH2 O N
OH OH

O
-

OH OH

CH2

O
O

CO2

LDH

+
C O + H

HO C H

CH3

CH3

pyruvate
(B)

L-lactate
(P)

NADH
(A)

O
+

OH OH
H

O
O
O

O
NH2

P
O CH2 O

(+)
N

OH OH

NAD+
(Q)

b) Derivation assuming rapid equilibria for substrate binding (Appendix C and Problem Set 3, question 4.d):

v i = k cat [EAB]
[E][A]
[EA][B]
and K'B =
KA =
[EA]
[EAB]
[E]o = [E] + [EA] + [EAB]

Vmax [B]
[A]
K'B + [B]
vi =
K AK'B
+ [A]
K'B + [B]

In this case, there is no [B] term in the numerator of the expression for KM(obs) because there is no EB
complex.
c) Experimental test for ordered or partially ordered ternary complex mechanisms - lines
intersecting above the x-axis in double reciprocal plots. There is no Ka[B] term in the expression
for KM(obs), and as a result, KM(obs) decreases from KA to 0 with increasing [B]. As before,
Vmax(obs) increases hyperbolically with increasing [B]. In the limiting case shown above, KM(obs)
decreases to 0 as [B]. For mechanisms in between random and ordered, KM(obs) either stays the
same or decreases with increasing [B].

Ternary Ordered

[B]1

increasing [B]

1/vi

[B]2

K M (obs)
decreasing

[B]3
[B]
Vmax(obs)increasing

1/[A]

Vmax [B]
[A]
KB + [B]
vi =
and again
K AK'B
+ [A]
K'B + [B]
K obs
1
1
1
M
= obs + obs
, where
vi
Vmax Vmax [A]

KM(obs) decreases with increasing [B]

since B facilitates the binding of A.
Note, Vmax(obs) still increases
hyperbolically with increasing [B].
B is required for catalysis and
facilitates the binding of A.

d). Product inhibition pattern for an ordered ternary complex mechanism using lactate
dehydrogenase as an example.
A = NADH Q = NAD
B = pyruvate P= lactate
A
E

If I = lactate, then at a fixed pyruvate concentration, a plot of

1/vi vs. 1/[NADH] at various [lactate] will exhibit
uncompetitive inhibition

B
EA

P
(EAB

EPQ)

EQ

Inhibitor
P
Q

plot
1/vi vs 1/[A]
1/vi vs 1/[A]

pattern
uncompetitive
competitive

fixed
B
B

This says that A binds first. (Note Q inhibition at a fixed A

exhibits a noncompetitive-like pattern when 1/vi vs. 1/[B]
plots are made. Q binds before B and the derivation is
complex. Don't worry about this issue.)

(ordered sequential mechanism)

C Binary Complex Mechanism ("ping-pong" or double displacement) - see Problem Set 3, question 5)
1. General scheme: there is no ternary complex where both A and B are present on the enzyme
surface at the same time. Rather, catalysis occurs by two binary complexes, EA and E*B.
E + A

EA

k1

KA
E* + B
KB

E* + P

k2

E*B

+ Q

The first binary complex forms a modified enzyme species, E*, and produces one product (i.e., vi = dP/dt
= k1[EA]). The second complex, E*B, regenerates the original enzyme form, E, and produces the second
product, Q. E* is the modified enzyme form, which contains a part of the initial substrate (i.e., electrons,
phosphate, amino group in the form of a Schiffs' base to vitamin B6, etc.). Cleland notation:
(1) Ping-pong or binary complex mechanism:
A
E

P
(EA

E*P)

B
E*

(2) Sequential-ternary complex mechanism

(E*B

EQ)

A
E

B
EA

P
(EAB

EPQ)

EQ

(a) Ordered: A first and then B

(b) Random: either A or B in the first step
2. Derivation assuming rapid equilibria for substrate binding and a steady state for the E*, E*B
intermediates (Problem Set 3, question 5.c somewhat like competitive inhibition because both substrates
compete for binding to the same site on the enzyme.):
Vmax [B]
[A]
v i = k1[EA] or k 2 [E * B]
K b + [B]
vi =
[E][A]
[E*][B]
Ka [B]
KA =
and K B =
+ [A]
[EA]
[E * B]
K b + [B]
, where
d([E*] + [E * B])
= 0 = k1[EA] k 2 [E * B]
k1k 2
dt
Vmax =
k1 + k 2
k2
[EA]
or
=
[E * B]
k1
kK
kK
Ka = 2 A ; Kb = 1 B
[E]o = [E] + [EA] + [E*] + [E * B]
k1 + k 2
k1 + k 2

In the binary complex mechanism case, there is no Kab term since a ternary EAB complex never
forms. KM(obs) increases with increasing [B] because B binds to enzyme tying it up in the E*B
complex, preventing A from binding until product is released.

3. Experimental test for a binary complex mechanism - parallel lines in double reciprocal
plots. There is no Kab term since EAB does not exist, and as a result, both Vmax(obs) and
KM(obs) depend on [B] by the same simple hyperbolic expression.
Vmax[B]
increasing
[A]
Binary complex (parallel lines)
[B]
[B]1
Kb + [B]
vi =
and again
K
[B]
a
[B]2
+ [A]
K b + [B]
[B]3
[B]

1/vi

K obs
1
1
1
M
= obs + obs
, where
vi
Vmax Vmax [A]

Vmax(obs) and KM(obs) show the

same dependence on [B] so the slope
in double reciprocal plots is
independent of [B]. Both Vmax(obs)
and KM(obs) increase with increasing
[B] because B is required to
regenerate E, but its binding to E*
prevents (competes with) the binding
of A to E.

K M (obs)
increasing

Vmax(obs)increasing

1/[A]

4. Examples of binary complex mechanisms (Problem Set 3, Question 5).

(a) Flavoprotein oxidases where E* = E(FADH2) -- as opposed to dehydrogenases
(b) Reactions with covalently modified enzyme intermediates. The classical example of this is
vitamin B6 (pyridoxal phosphate) - transaminase activity:
Transaminase example of a binary complex mechanism:
NH2

O
C
CO 2
H3 N C H

E Pi

R1

CH 2

CH2
OH

transaminase
E* Pi O

NH2

CH2

CO2
+

R2
B - keto acid(2)

E* Pi O

CH 2

C
OH

CH 3
N
Enzyme bound B6-NH2
(pyridoxamine-Pi)

CO 2
+

transaminase
E Pi

CH 2

O C
R1
P - keto acid(1)

CH 3
N
Enzyme bound B6 -NH2
(pyridoxamine-Pi)

N
CH 3
Enzyme bound B6
(pyridoxal-Pi)

A - amino acid(1)

OH

CH 2

H
CO2
OH

CH 3
N
Enzyme bound B6
(pyridoxal-Pi)

H 3N C H
R2
Q - amino acid(2)

C. Experimental tests for determining the mechanism of bisubstrate reactions (i.e. the purpose or
motivation of this lecture). Is the reaction catalyzed by a binary or ternary complex mechanism? If it
is a ternary mechanism, is the binding of substrates random or ordered?
1. Steady state kinetic patterns for 1/vi vs. 1/[A] plots at varying [B]
a.) Converging lines -- ternary complex (pp. 2-4).
i. Random - lines converge on the x-axis indicating that KM(obs) does not depend on [B]
ii. Ordered, A before B - KM(obs)0 with increasing [B] - line becomes parallel to the
1/[A] x-axis as [B] because 1/KM(obs) and slope (KM(obs)/VMAX(obs))0.
b.) Parallel lines -- indicates, but does not "prove" a binary complex mechanism (p. 6).
2. Product inhibition patterns for the ternary complex mechanism (idealized).
a.) 1/vi vs.
1/[A] at fixed
[B]

Inhibit with varying

concentrations of the
product from B.

noncompetitive inhibition
(only Vmax(obs) is decreased)

random addition
of A and B

b.) 1/vi vs.

1/[A] at fixed
[B]

Inhibit with varying

uncompetitive inhibition
concentrations of the
(both Vmax (obs) and KM(obs)
product from B to see if are decreased)
the product of B affects
the binding of A

ordered
addition; A first,
then B

3. Isolation of individual half reactions and the covalent enzyme intermediate indicates that a
binary complex mechanism occurs (i.e., pyridoxamine for transaminases -- p. 6).
4. Isotope exchange reactions can provide evidence for covalent enzyme intermediates and a
binary complex mechanism. (See next page.)
Classic example of exchange reactions: Sucrose phosphorylase (see other Biochemistry
Textbooks))
glucose-1-P + fructose
Overall reaction: sucrose
+
Pi
CH2OH

CH2OH

O
O

glucose-1-Pi
O

(E)
O

CH2OH

+
(-)O

OH
O(-)
CH2OH

CH2 OH

Pi

sucrose

Plots of 1/vi vs. 1/[sucrose]

at various [Pi] show parallel
lines. Both Vmax(obs) and
KM(obs) are increasing with
increasing [Pi].

P
O(-)

(-)O
OH

fructose
CH2 OH

Vmax [Pi]
[sucrose]
Kb + [Pi]
vi =
Ka [Pi]
+ [sucrose]
Kb + [Pi]

a. Examine for half reactions by isotope techniques (*radioactive label) - the first two work.
(i).

Fructose* + (glucose-fructose)

fructose*

+ sucrose*

sugar* = 14C

CH2 OH
CH2OH

OE

O
1st half
reaction
+

E-O-H

CH2OH

OH

CH2OH

CH2 OH

CH2 OH

fructose can exchange

into sucrose

There is a rapid incorporation of label into sucrose in the absence of Pi.

(ii).

Pi* + glucose-1-P
2nd half
reaction

CH2OH
O

OE

CH2O H
O
+

P* = 32P

Pi* + glucose-1-P*

E-O-H

P
(-)O

OH
O(-)

(-)O

O(-)

phosphate can exchange into glucose-1-Pi

There is a rapid incorporation of radiolabel into glucose-1-P in the absence of fructose

b. Mechanism: Two half reactions:
sucrose
+ E
E-glucosyl + fructose
glucose-1-P +

E-glucosyl + Pi,

This half reaction explains

fructose* exchange into sucrose.
This half reaction explains Pi*
exchange into glucose-1-P

c. Overall mechanism: two binary complexes with E-glucosyl intermediate

sucrose + E
E-glucosyl + fructose
E-glucosyl + Pi
E + glucose-1-P
d. This contrasts with maltose phosphorylase, which requires a ternary complex and exhibits no
exchange reactions, except in the presence of all substrates (and products).
Maltose + Pi + E

E (maltose, Pi)

E + glucose + glucose-1-P

In this case the following half reactions do not work (note: maltose = glucose-glucose).
(i).
Pi* + glucose-1-P
no exchange
(ii). glucose* + (glucose-glucose)
no exchange

General Rules for Exchange Reactions:

All reactions involve transfers and could be

catalyzed by binary complexes:
Substrate
that gets smaller

Substrate
that gets larger

X~Y + Z

X + Y~Z

Group that gets

transferred

fructose-glucose + HPO4=

fructose + glucose-1-Pi

Potential half reactions and exchanges:

X~Y + E

E~Y + X

E~Y + Z

+ Y~Z

Appendix A: Summary of Bisubstrate Reactions and Inhibition Patterns

(Equations for two different small molecules binding to an enzyme see also Appendix C)

10

A. Bisubstrate reactions: fix [B] and vary [A] and then see how Vmax(obs) and KM(obs) depend on [B].
vi =

obs
Vmax
[A]
obs
K M + [A]

Vmax [B]
[A]
K b + [B]
=
K ab + K a [B]
+ [A]
K b + [B]

1. Ternary complex mechanism: Kab 0, and KM(obs) depends on [B] to a lesser extent or not at
all compared to the hyperbolic dependence of Vmax(obs) on [B]. As a result, the slope of 1/vi versus
1/[A] varies with [B] and intersecting lines are observed. Remember, the slope in a double
reciprocal plot is KM(obs)/Vmax(obs). If KM(obs) shows no dependence on [B], the mechanism
involves random addition of substrates because the binding of B has no affect on the binding of A
in the steady state. If KM(obs) approaches 0 as [B] increases, then the mechanism involves an
ordered addition of substrates with A binding first.
2. Binary complex mechanism: Kab = 0 so that Vmax(obs) and KM(obs) increase with increasing [B]
and to the same extent (i.e. [B] / (Kb + [B])). As result, the slope of 1/vi versus 1/[A] does not vary
with [B], and parallel lines are observed in double reciprocal plots.
B. Inhibition Patterns (see Lecture 1- Olson): Fix [I] {and [B] if a bisubstrate reaction}, vary [A],
and then see how Vmax(obs) and KM(obs) depend on [I]. KI is the equilibrium dissociation constant
for I binding to free enzyme, E, and K'I is the equilibrium dissociation constant for I binding to the
enzyme substrate complex, EA.
Vmax

[A]
" [I] %
$$1 +
''
obs
Vmax
[A]
# K I! &
v i = obs
=
"
K M + [A]
[I] %
''
K M $$1 +
# KI &
+ [A]
"
[I ] %
$$1 +
'
K I! '&
#

1. Competitive
(I only binds to E.)
obs
Vmax
= Vmax
!
[I] $
#
&&
=
K
1
+
K obs
M
M#
" KI %

2. Uncompetitive
(I only binds to EA.)
obs
Vmax
=

K obs
M =

Vmax
" [I] %
$$1 +
''
# K I! &
KM
" [I] %
$$1 +
''
# K I! &

3. Noncompetitive
(I binds equally well to E
and EA so K'I = KI.)
obs
Vmax
=

Vmax
!
[I] $
##1 +
&&
" KI %

K obs
M = KM

11
Appendix B: Wendel/Simonak Crib Sheet for Remembering Enzyme Mechanisms and Inhibition
Patterns:
obs
Vmax

Vmax [B]
[A]
obs
K b + [B]
Vmax [A]
v i = obs
=
K ab + K a [B]
K M + [A]
+ [A]
K b + [B]

[A]
" [I] %
$$1 +
''
# K I! &
vi =
"
[I] %
$
''
K obs
M $1 +
# KI &
+ [A]
"
[I ] %
$$1 +
'
K I! '&
#

BROCNU Effects on KM's for mechanisms: increase, none, decrease (KM's up, none, down).
Letter
KM(obs) Vmax(obs)
Key parameters
B

Binary Complex

Kab = 0

Random ternary

Kab = KaM#b

Ordered ternary

Ka[B] = 0

Competitive inhibition

KM(1+[I]/KI)

Noncompetitive inhibition

KM

Uncompetitive inhibition

KM/(1+[I]/K'I)

Appendix C: General Rapid Equilibrium Mechanism and Equations: Supplemental material (Not
required)
A

EA +
B
KA

K'B

kcat
EAB

P, Q

+
B
KB

K'A

A
EB

vi = kcat[EAB]

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

[E][A]
[EA]
K [EA]
[E] = A
[A]
KA =

KB =

[E][B]
[EB]

[EB] =

[E][B]
KB

[EA][B]
[EAB]
K! [EAB]
[EA] = B
[B]

K!B =

K A K!B [EAB]
K A K!B [EAB]
[B] K A K!B [EAB]
[EB] =

=
[A][B]
[A][B]
K B [A]
KB
" K K! K K! K!
%
[E]total
[E]total = $$ A B + A B + B + 1''[EAB] [EAB] =
%
"
K K! K K! K!
# [A][B] K B [A] [B] &
$$ A B + A B + B + 1''
# [A][B] K B [A] [B] &
[E] =

v i = k cat [EAB] =

k cat [E]total

k cat E total [A][B]

[A][B]
=
% [A][B]
" K K! K K! K!
K K!
K A K!B + A B [B] + K!B [A] + [A][B]
$$ A B + A B + B + 1''
KB
# [A][B] K B [A] [B] &
*

Factor out [A] in the denominator and then divide numerator and denominator by (K'B + [B]):

k cat E total [B]

[A]
K!B + [B]
vi =
K K!
K A K!B + A B [B]
KB
+ [A]
K!B + [B]

obs
VMAX
=

k cat E total [B]

K!B + [B]

K obs
=
M

K A K!B
[B]
KB
K!B + [B]

K A K!B +

12

1. When [A] is varied at several different "fixed' [B]:

k catE total [B]
[A]
k E
[B]
KB + [B]
obs
VMAX
= cat total
vi =
K K
KB + [B]
K AKB + A B [B]
KB
+ [A]
KB + [B]

13

Kobs
M =

K AKB
[B]
KB
KB + [B]

K AKB +

a. Ordered Mechanism: KB

Kobs
M for A =

K AKB
; decreases to 0 as [B] .
KB + [B]

b. Random Mechanism: KB = KB; KA = KA

K AKB + K A [B]
K (K + [B])
= A B
= K A ; unaffected by [B]
KB + [B]
KB + [B]

Kobs
M for A =

c. Competition between A and B:

KB > KB

Kobs
M for [A] =

K AKB
[B]
KB
KB + [B]

K AKB +

When KB

[B]
Kobs
; increases with [B], somewhat
M for A = K A 1 +
KB
like a binary complex mechanism. However, in this case,
KM(obs) goes to infinity with a function that looks like
competitive inhibition, with B as an inhibitor.

k catE total [A]

[B]
K AKB
+ [A]
KB
2. The opposite situation: [B] is varied at various "fixed" [A]: v i =
KBK A + KB[A]
+ [B]
K AKB
+ [A]
KB
a. Ordered Mechanism: KB
Kobs
M for B =

KB (K A + [A])
; decreases from infinitely large to K'B
[A]

b. Random Mechanism: KB = KB; KA = KA

K obs
M for B =

KBK A + KB[A] KB (K A + [A])

=
= KB = K B ; unaffected by [A]
K A + [A]
K A + [A]

c. Competition between A and B:

KB > KB
K obs
M for B =

KB K A + [A]

K A KB
+ [A]
KB

When KB
! [A] $
#
&&
K obs
M for B = K B #1+
" KA %

; increases with [A] like a

binary complex mechanism, but again in this case KM(obs)

goes to infinity like competitive inhibition with increasing [A].