1

BIOS. 301 - Lecture 3 - Olson - More Complex Kinetic Patterns

(LP0B-6, pp.207-208; 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]
vi = , where the K's are referred to as "Michaelis" constants. Rearranging:
Kab + Ka [B] + Kb [A] + [A][B]

Vmax[B] obs
Vmax [A]
[A] vi = ;
Kb + [B] Kobs
vi = M + [A]
Kab + Ka [B]
+ [A] obs Vmax[B] Kab + Ka [B]
K b + [B] Vmax = ; Kobs
M =
Kb + [B] Kb + [B]

Analysis: Fix B and vary A and then Remember the reciprocal form is:
repeat the experiment at another [B] to
1 1 Kobs
M 1
see how KM(obs) and Vmax(obs) depend on = obs + obs
the concentration of the second substrate. vi Vmax Vmax [A]

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 prior, rapid equilibrium scheme:

For this mechanism, the enzyme is acting like a

template rather than a donor or acceptor.
A B
EA
KA kcat
E K'B Release of products P and Q
B A EAB EPQ

KB EB K'A

Kab
[E][A] [E][B] [E][A][B]
KA = ; 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
 2
ii.) The kinetic characteristics of ternary complex mechanisms are converging lines in
double reciprocal plots. This means that the Kab has a finite value and that the species
EAB exists. Experimentally, [B] affects the slope of a 1/vi vs/ 1/[A] plot.

Vmax[B] B1
[A]
Kb + [B]
v i = K + K [B] and
ab a + [A] 1/vi
K b + [B] B2
1 1 Kobs
M 1 Converging
= + , lines
vi obs obs [A]
Vmax Vmax
B3
where Vmax(obs) and KM(obs) show different increasing
dependencies on [B]. [B]

This test holds whether or not the rapid

equilibrium assumption is valid.

1/[A]
- 1/KM (obs) 1/Vmax (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 • KB. (Problem Set 3, question 3 – like noncompetive inhibition)

a) Examples: most kinases, including creatine kinase

NH NH
H2 H2 O
C C C C
O N O N (-)
C
NH2
C N P O
+ ATP CH3 H O + ADP
O CH3 (-) O
(-) B (-) Q
Creatine Creatine-Pi
A P
b) Derivation from mechanism assuming rapid equilibria for substrate binding and the scheme
on page 1. (see Appendix C, Problem Set 3, question 3.d)
Vmax [B] Vmax [B]
v i = k cat [EAB] [A] [A]
K B + [B] K B + [B]
[E][A] [EB][A] vi = =
KA = = K"A = K AK B + K A [B] K A (K B + [B])
[EA] [EAB] + [A] + [A]
K B + [B] K B + [B]
[E][B] [EA][B] or
KB = = K"B =
[EB] [EAB]
Vmax [B]
[E]o = [E] + [EA] + [EB] + [EAB] [A]
K B + [B]
vi = ; note K ab = K AK B
K A + [A]

!
!
 3
c) Experimental test for a random ternary complex mechanism - lines intersecting on the x-
axis 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])
Vmax[B]
[A]
Ternary Random
[B]1 K B + [B]
increasing [B] vi =
[B]2 Remember : K A + [A] and

[B]3 1 1 Kobs
M 1
= +
vi obs obs [A]
Vmax Vmax
1/vi

[B]!"

where KM(obs) shows the no dependence on

K M (obs) [B] and the lines intersect on the x-axis.
unchanging Vmax(obs) does increase and, because
Vmax(obs)increasing
KM(obs) is unchanged, the slope decreases
with increasing [B]. Bottom line - B is
required for catalysis but does not affect the
1/[A] binding of 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 plot pattern
creatine + ATP creatine-Pi + ADP Q B 1/vi vs. 1/[A] noncompetitive
[A] [B] [P] [Q] P B 1/vi vs. 1/[A] competitive
P A 1/vi vs. 1/[B] competitive*
The non-competitive inhibition pattern suggests that Q A 1/vi vs. 1/[B] competitive
the binding of A and B is not ordered but random.
*Since the Pi of creatine-Pi overlaps the γ-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 EAB EPQ release of products
KA 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 N
N

N N N
N CH2
CH2 O O
O O

OH OH CO2 -
CO2 - P OH OH
P LDH O O
O O C O + H
+ O
H H O + HO C H +
O
H
O CH3 CH3
NH2 O NH2
O pyruvate P (+)
P L-lactate
(B) (P) O N
O O CH2 N O CH2
O O
NADH NAD+
(A) OH OH (Q)
OH OH
b) Derivation from mechanism assuming rapid equilibria for substrate binding (Appendix C and Problem
Set 3, question 4.d):

v i = k cat [EAB] Vmax [B]

[A]
[E][A] [EA][B] K'B + [B]
KA = and K'B = vi =
[EA] [EAB] K AK'B
+ [A]
[E]o = [E] + [EA] + [EAB] 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]. At [B]→∞, KM(obs) varies between KA (random) and 0
(ordered).

Ternary Ordered Vmax [B]

[B]1 [A]
increasing [B] K"B + [B]
vi = and again
K AK'B
+ [A]
K'B + [B]
1 1 Kobs
M 1 , where
[B]2 = obs + obs
vi Vmax Vmax [A]
1/vi

!
K M (obs) KM(obs) decreases with increasing [B]
decreasing since B facilitates the binding of A.
[B]3 Note, Vmax(obs) still increases
hyperbolically with increasing [B].
[B]!"
Vmax(obs)increasing
B is required for catalysis and
facilitates the binding of A.
1/[A]
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d). Product inhibition pattern for an ordered ternary complex mechanism using lactate
dehydrogenase as an example.
A = NADH Q = NAD If I = lactate, then at a fixed pyruvate concentration, a plot of
B = pyruvate P= lactate 1/vi vs. 1/[NADH] at various [lactate] will exhibit
uncompetitive inhibition
A B P Q Inhibitor plot pattern fixed
P 1/vi vs 1/[A] uncompetitive B
E EA (EAB EPQ) EQ E Q 1/vi vs 1/[A] competitive B

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

(ordered sequential mechanism) exhibits a noncompetitive pattern when 1/vi vs. 1/[B] plots
are made)

C Binary Complex Mechanism (other names: "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.
k1
E + A EA E* + P
KA
k2
E* + B E*B E + Q
KB
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 B6, etc.). Cleland notation:

(1) Ping-pong or binary complex mechanism: (2) Sequential-ternary complex mechanism

A P B Q A B P Q

E (EA E*P) E* (E*B EQ) E E EA (EAB EPQ) EQ E

(a) Ordered: A first and then B

(b) Random: either A or B in the first step
2. Derivation from mechanism assuming rapid equilibria for substrate binding and a steady state for
the E*, E*B intermediates (Problem Set 3, question 5.c – like competitive inhibition since both substrates
compete for binding to the same site on the enzyme):
Vmax [B]
v i = k1[EA] or k 2 [E * B] [A]
K b + [B]
[E][A] [E*][B] vi = 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 =
k2 [EA] k1 + k 2
or = k 2K A k1K B
k1 [E * B] Ka = ; Kb =
k1 + k 2 k1 + k 2
[E]o = [E] + [EA] + [E*] + [E * B]

!
!
 6
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 which prevents A from binding until product is formed.

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.
increasing Vmax[B]
Binary complex (parallel lines) [B]
[A]
[B]1 Kb + [B]
vi = and again
[B]2
K a [B]
+ [A]
K b + [B]
[B]3
[B]!" 1 1 Kobs
M 1 , where
= +
vi obs obs [A]
Vmax Vmax
1/vi

Vmax(obs) and KM(obs) show the

K M (obs)
same dependence on [B] so the slope
increasing
in double reciprocal plots is
independent of [B]. Both Vmax(obs)
and KM(obs) increase with increasing
Vmax(obs)increasing [B] since B is required to regenerate
E, but its binding to E* prevents the
binding of A to E.
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 - transaminase activity:
Transaminase example of a binary complex mechanism:
NH2
O H
C CH2
transaminase CO 2
CO 2 CH 2 OH CH 2 OH
O E* Pi O + O C
H3 N C H + E Pi
R1
R1 N CH 3 N CH 3
P - ! keto acid(1)
A - ! amino acid(1) Enzyme bound B6 Enzyme bound B6 -NH2
(pyridoxal-Pi) (pyridoxamine-Pi)

NH2 O H
CH2 C CO2
CO2 CH 2 OH transaminase CH 2 OH + H 3N C H
O C + E* Pi O E Pi
O
R2
R2
N CH 3 N CH 3 Q - ! amino acid(2)
B - ! keto acid(2)
Enzyme bound B6-NH2 Enzyme bound B6
(pyridoxamine-Pi) (pyridoxal-Pi)
 7
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
a ternary mechanism, is it 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 Inhibit with varying uncompetitive inhibition ordered addition;

1/[A] at fixed concentrations of the obs obs
( K M , V max are decreased) A first, then B
[B] product from B to see if
the product of B affects
the binding of A

b.) 1/vi vs Inhibit with varying noncompetitive inhibition random addition

1/[A] at fixed concentrations of the obs
(only V max decreased) of A and B
[B] product from B.

3. Isolation of individual half reactions and the covalent enzyme intermediate indicates (if
not proves) 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))
Overall reaction: sucrose + Pi glucose-1-P + fructose
CH2OH
CH2OH
O glucose-1-Pi
O
O
O (E)
O P
CH2OH O + P O(-)
(-)O OH + (-)O
O(-)
CH2OH OH
CH2 OH Pi
fructose
sucrose
CH2 OH

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

Vmax [Pi]
at various [Pi] show parallel [sucrose]
lines. Both Vmax(obs) and Kb + [Pi]
vi =
KM(obs) are increasing with Ka [Pi]
+ [sucrose]
increasing [Pi]. Kb + [Pi]

a. Examine for half reactions by isotope techniques (*radioactive label) - the first two work.
 8
(i). Fructose* + (glucose-fructose) fructose* + sucrose* sugar* = 14C
CH2 OH
CH2OH OE
O
O
1st half
reaction
+
CH2OH O + E-O-H CH2OH OH

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 Pi* + glucose-1-P* P* = 32P

CH2O H
CH2OH 2nd half
OE reaction O
O
+
O
O
+ E-O-H
P
(-)O OH O P
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 This half reaction explains
fructose* exchange into sucrose.
glucose-1-P + E E-glucosyl + Pi, 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
 9
Appendix A: Summary of Bisubstrate Reactions and Inhibition Patterns
(Equations for two different small molecules binding to an enzyme –Required see also Appendix C)

A. Bisubstrate reactions: fix [B] and vary [A] and then see how Vmax(obs) and KM(obs) depend on [B].
Vmax[B]
obs [A]
Vmax [A] Kb + [B]
vi = =
Kobs
M + [A]
K ab + Ka [B]
+ [A]
Kb + [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). Note that 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 order addition of substrates with A binding first.
2. Binary complex mechanism: Kab = 0 so that Vmax(obs) and KM(obs) increase with increasing
[B] 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} and 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] %
$$ 1 + '
obs
Vmax [A] # KI! '&
v i = obs =
K M + [A] " [I] %
K M $$ 1 + '
# K I '&
+ [A]
" [I ] %
$$ 1 + '
# K !I '&

1. Competitive 2. Uncompetitive 3. Noncompetitive

(I only binds to E.) (I only binds to EA.) (I binds equally well to E
obs obs Vmax and EA so K'I = KI.)
Vmax = Vmax Vmax = " % Vmax
$$ 1 + [I] ''
" obs
[I] % Vmax =
K obs
M = K M $ 1 + ' # K I! & " [I] %
# KI & $1 + '
KM # KI &
Kobs
M = "
[I] % K obs
M = KM
$$ 1 + '
# K I! '&
!

!
 10
Appendix B: Wendel/Simonak Crib Sheet for Remembering Enzyme Mechanisms and Inhibition
Patterns:

obs
Vmax
[A]
Vmax[B] # [I] &
obs [A] %1 + (
Vmax [A] Kb + [B] $ KI" '
vi = = vi =
Kobs
M + [A]
K ab + Ka [B] K obs
# [I] &
+ [A] M %1 + (
Kb + [B] $ KI '
+ [A]
# [I ] &
%1 + (
$ KI" '

BROCNU Effects on KM's for mechanisms: increase, none, decrease (KM's up, none, down).
Letter K!M(obs) Vmax(obs) Key parameters
B Binary Complex ↑ ↑ Kab = 0
R Random ternary ⇔ ↑ Kab = Ka•Kb
O Ordered ternary ↓ ↑ Ka[B] = 0
C Competitive inhibition ↑ ⇔ KM(1+[I]/KI)
N Noncompetitive inhibition ⇔ ↓ KM
U Uncompetitive inhibition ↓ ↓ KM/(1+[I]/K'I)
 11
Appendix C: General Rapid Equilibrium Mechanism and Equations: Supplemental material (Not
required)
EA +
KA B
A
+ K'B kcat
vi = kcat[EAB]
E EAB P, Q
+
B K'A
A
KB +
EB

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

[E][A] [E][B] [EA][B]

KA = KB = K"B =
[EA] [EB] [EAB]

K A [EA] [E][B] K"B[EAB]

[E] = [EB] = [EA] =
[A] KB [B]

K AK"B[EAB] [B] K AK"B[EAB] K K" [EAB]

[E] = [EB] = • = A B
[A][B] KB [A][B] K B[A]

# K K" K K" K" &

[E]total = % A B + A B + B + 1([EAB]
$ [A][B] K B[A] [B] '

k catE total [A][B]

v i = k catE total =
K K"
! K AK"B + A B [B] + K"B[A] + [A][B]
KB

k catE total [B] K AK"B

[A] K AK"B + [B]
! K"B + [B] obs k catE total [B] KB
vi = VMAX = Kobs
M =
K K" K"B + [B] K"B + [B]
K AK"B + A B [B]
KB
+ [A]
K"B + [B]

!
 12
1. When [A] is varied at several different "fixed' [B]:
k catE total [B] K AK"B
[A] K AK"B + [B]
K"B + [B] obs k E [B] KB
vi = VMAX = cat total Kobs
M =
K K" K"B + [B] K"B + [B]
K AK"B + A B [B]
KB
+ [A]
K"B + [B]

a. Ordered Mechanism: KB → ∞
K AK"B
! Kobs
M for A = ; decreases to 0 and [B] → ∞.
K"B + [B]

b. Random Mechanism: KB = K’B; KA = K’A

K AK"B + K A [B] K (K" + [B])
! Kobs
M for A = = A B = K A ; unaffected by [B]
K"B + [B] K"B + [B]
c. Competition between A and B:
K’B > KB When ⇒ K’B → ∞
!
K AK"B " [B] %
K AK"B + [B] Kobs
M for A = K A $1 + ' ; increases with [B] somewhat
KB # KB &
Kobs
M for [A] =
K"B + [B] like a binary complex mechanism, but in this case to infinity
with a function that looks like competitive inhibition with B
as an inhibitor.
!
! k catE total [A]
[B]
K AK"B
+ [A]
KB
2. The opposite situation: [B] is varied at various "fixed" [A]: v i =
K"BK A + K"B[A]
+ [B]
K AK"B
+ [A]
KB
a. Ordered Mechanism: KB → ∞
K"B (K A + [A])
Kobs
M for B = ; decreases from infinitely large to K'B
[A]
!
b. Random Mechanism: KB = K’B; KA = K’A
K"BK A + K"B[A] K" (K + [A])
Kobs
M for A = = B A = K"B = K B; unaffected by [A]
! K A + [A] K A + [A]
c. Competition between A and B:
K’B > KB When ⇒ K’B → ∞
!
K"B (K A + [A]) " [A] %
Kobs
M for [A] = Kobs
M for A = K B $1 + ' ; increases with [A] like a
K AK"B # KA &
+ [A]
KB binary complex mechanism but in this case to infinity like
competitive inhibition.
!
!