II.

Kinetics
Reaction velocities and rate laws
6.1 Express the reaction velocities of the following reactions in terms of
each reactant/product:
(1) S → P
(2) E + S → E + P
(3) ATP + H2O → ADP + Pi
(4) NAD+ + H+ (+ 2 e–) → NADH
(5) H2O → H+ + OH–
(6) 2M → D
Answer:
(1) v = –d[S]/dt = d[P]/dt
(2) v = –d[S]/dt = d[P]/dt, d[E]/dt = 0
(3) v = –d[ATP]/dt = –d[H2O]/dt = d[ADP]/dt = d[Pi]/dt
(4) v = –d[NAD+]/dt = –d[H+]/dt = d[NADH]/dt
(5) v = –d[H2O]/dt = d[H+]/dt = d[OH–]/dt
(6) v = –½∙d[M]/dt = d[D]/dt (two moles of M react to one mol of D, and
[M] changes twice as fast as
[D]).
6.2 What is the reaction velocity for a reaction of type aA + bB → cC +
dD?
Answer: v = –(1/a)∙d[A]/dt = –(1/b)∙d[B]/dt = (1/c)∙d[C]/dt =
(1/d)∙d[D]/dt
6.3 Write the rate laws for the forward and reverse reactions:
(1) S ⇄ P
(2) E + S ⇄ E + P
(3) ATP + H2O ⇄ ADP + Pi
38
(4) NAD+ + H+ (+ 2 e–)⇄ NADH
(5) H2O ⇄ H+ + OH–
(6) 2M ⇄ D
The forward rate constant is kf, the rate constant for the reverse
reaction is kr.
Answer:
(1) vf = kf∙[S], vr = kr∙[P]
(2) vf = kf∙[S][E], vr = kr∙[P][E]
(3) vf = kf∙[ATP][H2O], vr = kr∙[ADP][Pi]
(4) vf = kf∙[NAD+][H2O], vr = kr∙[NADH][H+]
(5) vf = kf∙[H2O], vr = kr∙[H+][OH–]
(6) vf = kf∙[M]2, vr = kr∙[D]
6.4 What is the order of the reaction of iodine and hydrogen with
respect to the reactants, and
what is the overall order of the reaction? What is the molecularity?
What are the corresponding
orders and molecularities for the individual steps of the reaction of
bromine with hydrogen?
Answer: For the iodine reaction the order is one with respect to I2, one
with respect to H2, and two
overall. The molecularity is two. For the bromine reaction, the order is
one with respect to Br2 and
one overall for the starting reaction. The molecularity is one. For the
reaction of bromine radicals
with H2, the order is one with respect to Br∙ and with respect to H2,
and two overall. The molecularity
is two. The reaction of bromine with a hydrogen radical is a first-order
reaction with respect to Br2
and first order with respect to H∙; the overall reaction is second order.
The molecularity is two. The
recombination of bromine radicals is second order with respect to Br∙
and overall, the molecularity is
two.
39
6.5 The concentration of a reactant A decays exponentially according to
A(t) = A0e–kt with the
rate constant k. What is the reaction velocity as a function of time?
What is the initial velocity of the
reaction?
Answer: The reaction velocity is defined as
𝑝𝑝 = -
𝑝𝑝[𝐴𝐴]
𝑝𝑝𝑝𝑝
The first derivative of the concentration is
𝑝𝑝[𝐴𝐴]
𝑝𝑝𝑝𝑝 = -𝑘𝑘𝐴𝐴0𝑟𝑟-𝑘𝑘𝑤𝑤
which gives
𝑝𝑝 = 𝑘𝑘𝐴𝐴0𝑟𝑟-𝑘𝑘𝑤𝑤
The velocity thus decreases exponentially from the initial value of v =
kA0 to zero.
40
Integrated rate laws for uni- and bimolecular reactions
7.1 You measure the concentration of a reactant as a function of time
and obtain the following
dataset:
t (s) c(t) (µM)
0 10
1 7.1
2 5.0
3 3.5
4 2.5
5 1.7
6 1.3
8 0.6
10 0.3
What are the reaction order and the rate constant?
Answer: The starting concentration is halved after 2 s (t½, c = c0/2), and
halved a second time after 4 s
(c = c0/4), indicating that t½ is constant. After 6 s (3∙t½), c = c0/8, and
after 8 s (4∙t½), c = c0/16.
The half-life is t½ = 2 s, and the rate constant is k = ln2/t½ = 0.7/2 s =
0.35 s–1.
7.2 A reactant shows the following decrease in concentration with
reaction time:
t (s) c(t) (µM)
0 1
1 0.74
2 0.59
3 0.49
4 0.42
5 0.37
6 0.32
8 0.27
10 0.22
Determine the reaction order and estimate the rate constant.
41
Answer: The first half-life is t½,1 = 3 s, the second half-life is t½,2 = 5 s,
and hence the reaction must be
second order. The relation between half-life and rate constant is t½ =
(2kA0)–1, which gives
𝑘𝑘 =
1
2 ∙ 𝑝𝑝½,1 ∙ 𝐴𝐴0 = 0.166 𝜇𝜇𝑀𝑀-1 𝑝𝑝-1
𝑘𝑘 =
1
2 ∙ 𝑝𝑝½,2 ∙ 𝐴𝐴0 = 0.1 𝜇𝜇𝑀𝑀-1 𝑝𝑝-1
The rate constant is somewhere between 0.33 μM–1 s–1 and 0.4 μM–1
s–1.
7.3 An old tree trunk is discovered and submitted to radiocarbon
dating. The 14C percentage in
the analyzed samples is 10–12%. What is the age of the tree? [14C]0 =
10–10%, t½ = 5730 a.
Answer: The fraction of 14C is
�14𝐶𝐶(𝑝𝑝)� = �14𝐶𝐶�0𝑟𝑟-𝑘𝑘𝑤𝑤 = �14𝐶𝐶�0𝑟𝑟-
𝑓𝑓𝑓𝑓2
𝑤𝑤1/2𝑤𝑤
which can be rearranged to
𝑝𝑝 = -
𝑝𝑝1/2
𝑚𝑚𝑟𝑟2 ∙ 𝑚𝑚𝑟𝑟
�14𝐶𝐶(𝑝𝑝)�
�14𝐶𝐶�0 ≈ 38070 𝑔𝑔𝑟𝑟𝑃𝑃𝑝𝑝𝑝𝑝
7.4 You determine the concentration of a reactant that reacts on its
own at time t = 3.6 s as
c(t) = 0.27 mM. The starting concentration was 10 mM. Does the
reaction follow the rate law for
first- and second-order reactions? What is the rate constant?
Answer: The integrated rate law for a first-order reaction is
𝐴𝐴(𝑝𝑝) = 𝐴𝐴0 ∙ 𝑟𝑟-𝑘𝑘𝑤𝑤
A(3.6 s) = 0.27 mM, which gives k = 1.0 s–1. The integrated rate law for
a second-order reaction is
A(t) = A0/(1+ktA0), and A(3.6 s) = 0.27 mM gives k = 1.0 s–1. The single
data point is consistent with
both rate laws (the concentration curves intersect at one time point, in
this case for t = 3.6 s). To
deduce the rate law and reaction order, a series of concentrations at
different time points is
required.
42
7.5 The reactants A and B react to products in an irreversible reaction.
What is the concentration
of A and B at t = 5 s for (1) A0 = 1 µM, B0 = 10 µM, (2) A0 = 10 µM, B0 =
1 µM, (3) A0 = 6 µM, B0 = 5 µM
and for (4) A0 = 20 µM, B0 = 29 µM? k = 0.2 μM–1 s–1.
Answer: The concentrations are calculated from the integrated rate law
for the corresponding
second-order reaction as (1) A(t) = 0.38 µM, B(t) = 9.38 µM (product x =
0.62 µM); (2) A(t) = 9.38 µM,
B(t) = 0.38 µM (product x = 0.62 µM); (3) A(t) = 4.07 µM, B(t) = 3.07 µM
(product x = 1.93 µM); (4)
A(t) = 3.5 µM, B(t) = 12.5 µM (product x = 16.5 µM).
Comparison of (1) and (2): With the same difference |A0–B0|, reaction
velocity and amount of
product formed are identical.
Comparison of (2) and (3): These conditions have the same sum of
concentrations A0+B0, but A0 and
B0 are more similar in (3), thus |A0–B0| is smaller, and the reaction
velocity and amount of product
are increased.
Comparison of (2) and (4): The larger sum of concentrations A0+B0 in
(4) leads to a higher reaction
velocity and amount of product.
7.6 Derive the integrated rate law for the trimolecular reaction 3 A →
products. The rate
constant is k. What is a suitable plot to determine the rate constant by
linear regression?
Answer: The rate law is
-
𝑝𝑝[𝐴𝐴]
𝑝𝑝𝑝𝑝 = 3𝑘𝑘[𝐴𝐴]3
Separation of variables and integration from A0 to [A](t) gives
-
1
2�[𝐴𝐴](𝑝𝑝)�2 +
1
2𝐴𝐴
20
= -3𝑘𝑘𝑝𝑝
or
-
1
�[𝐴𝐴](𝑝𝑝)�2 +
1 2𝐴𝐴0
= -6𝑘𝑘𝑝𝑝
43
1/A2(t) is a linear function of time, and from the slope –6k of the linear
plot , the rate constant can be
obtained. Rearrangement gives the integrated rate law
[𝐴𝐴](𝑝𝑝) =
𝐴𝐴0
�1 + 6𝑘𝑘𝑝𝑝𝐴𝐴20
44
Reaction types
8.1 A reversible reaction A + B ⇄ AB can be monitored by fluorescence
because the fluorescence
of B increases upon AB formation. Binding is too fast to be measured
and already complete within
the dead time of your experiment. How can you determine Keq, k–1,
and k1?
Answer: Measure Kd in a fluorescence equilibrium titration of
fluorescent B with A, and k–1 in a
displacement reaction by adding an excess of non-fluorescent B to AB
and following the decrease in
fluorescence as a function of time. Calculate k1 as Keq∙k–1.
8.2 You start from a reactant A and monitor formation of the product B
of a reaction as a
function of time. B(t) can be described by a single exponential function
with a rate constant
k = 0.4 s–1. The end concentration of B is 7.5 µM. What are the possible
explanations for this behavior
and what is the meaning of the measured rate constant in each case?
Which piece of information is
needed to distinguish between the different possibilities?
Answer: (1) A reacts to B with a rate constant of 0.4 s–1. (2) A reacts
not only to B but can also react
to a different product C. k = kB + kC. (3) A reacts to B in a reversible
reaction with Keq = k–1/k1, k1 + k–1.
Distinction between (1) or (2), (3): (1) gives B∞ = A0, (2) and (3) give
B∞ < A0.
Distinction between (2) and (3): Analysis if other products than B are
formed (2) or testing if reaction
from B leads to formation of A (3).
45
8.3 An enzyme binds and hydrolyzes ATP. You want to populate the
enzyme-ATP complex to
study its binding to an interaction partner. ATP binding induces a
conformational change in the
enzyme, and the overall binding reaction is slow with kbind = 0.1 s–1.
The rate constant of ATP
hydrolysis is khyd = 10–3 s–1. How long do you have to incubate the
enzyme with ATP to maximize the
concentration of the ATP-bound form?
Answer: ATP binding and hydrolysis are consecutive reactions. The
maximum concentration of the
complex is achieved at the time t for which the first derivative of the
intermediate B(t) is zero, i.e.
𝑘𝑘𝑚𝑚
𝑎𝑎𝑓𝑓𝑢
�𝑟𝑟-
𝑢
[𝐵𝐵]( 𝑘𝑘𝑏𝑏𝑏𝑏𝑏𝑏𝑏
𝑘𝑘ℎ𝑦𝑦
𝑝𝑝) = 𝑏𝑤𝑤 - 𝑟𝑟-
𝑢𝑢 -
𝐴𝐴0 𝑘𝑘ℎ𝑦𝑦𝑏𝑏𝑤
𝑘𝑘𝑚𝑚
𝑤�
𝑎𝑎𝑓𝑓𝑢
𝑢
and hence
-𝑘𝑘𝑚𝑚𝑎𝑎𝑓𝑓𝑢𝑢𝑟𝑟-𝑘𝑘𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑤𝑤 - -𝑘𝑘ℎ𝑦𝑦𝑢𝑢𝑟𝑟-𝑘𝑘ℎ𝑦𝑦𝑏𝑏𝑤𝑤 = 0
Rearranging gives
𝑝𝑝 =
𝑚𝑚𝑟𝑟 𝑘𝑘𝑚𝑚𝑎𝑎𝑓𝑓𝑢𝑢
𝑘𝑘ℎ𝑦𝑦𝑢𝑢
𝑘𝑘𝑚𝑚𝑎𝑎𝑓𝑓𝑢𝑢 - 𝑘𝑘ℎ𝑦𝑦𝑢𝑢 = 46 𝑝𝑝
46
Rate-limiting steps
9.1 DNA strands (A, B) associate rapidly to mismatched duplexes (I;
kA,B→I) that readily dissociate
(kI→A,B) or slowly rearrange to the base-paired duplex (P) with kI→P.
What is the reaction velocity for
duplex formation as a function of the concentration of the DNA strands
A and B and what is the
apparent rate constant of duplex formation?
Answer: The reaction scheme is
The overall reaction velocity is
𝑝𝑝 = 𝑘𝑘𝐼𝐼→𝑃𝑃 ∙ [𝐼𝐼]
because this is the rate-limiting step. With
𝐾𝐾𝑢𝑢1 =
𝑘𝑘𝐼𝐼→𝐴𝐴,𝐵𝐵
𝑘𝑘𝐴𝐴,𝐵𝐵→𝐼𝐼 =
[𝐴𝐴][𝐵𝐵]
[𝐼𝐼]
we obtain
𝑝𝑝 = 𝑘𝑘𝐼𝐼→𝑃𝑃 ∙ [𝐼𝐼] =
𝑘𝑘𝐴𝐴,𝐵𝐵→𝐼𝐼𝑘𝑘𝐼𝐼→𝑃𝑃
𝑘𝑘𝐼𝐼→𝐴𝐴,𝐵𝐵 ∙ [𝐴𝐴][𝐵𝐵]
with
𝑘𝑘
𝑤𝑤𝑝𝑝𝑝𝑝 =
𝑘𝑘𝐴𝐴,𝐵𝐵→𝐼𝐼𝑘𝑘𝐼𝐼→𝑃𝑃
𝑘𝑘𝐼𝐼→𝐴𝐴,𝐵𝐵
47
Binding reactions: one step- and two-step binding
10.1 The binding of a ligand L to its interaction partner shows a
hyperbolic dependence on the
ligand concentration under pseudo-first order conditions. kobs as a
function of [L] gives Kd1 = 100 µM
and k2 = 1 s–1. The y-axis intercept is not well-defined and k–2 cannot
be determined accurately from
the fit – why? In an equilibrium titration, the overall dissociation
constant is determined to
Kd,overall = 10 nM. Calculate k–2. How can k–2 be measured directly
and why is the experimental
determination of k–2 difficult?
Answer: When k–2 is very small, and the intercept is not well-
determined and can be extracted from
the data with great uncertainty. k–2 can be calculated from the given
constants: The overall
equilibrium constant is
𝐾𝐾𝑢𝑢,𝑓𝑓𝑜𝑜𝑤𝑤𝑤𝑤𝑤𝑤𝑓𝑓𝑓𝑓 =
𝐾𝐾𝑢𝑢1𝑘𝑘-2
𝑘𝑘2
which gives
𝑘𝑘
-2 = 𝐾𝐾𝑢𝑢,𝑓𝑓𝑜𝑜𝑤𝑤𝑤𝑤𝑤𝑤𝑓𝑓𝑓𝑓
𝑘𝑘2
𝐾𝐾𝑢𝑢1 = 1 ∙ 10-4𝑝𝑝-1
k–
2 can be determined directly by adding an excess of unlabeled ligand to
a preformed complex of
protein and fluorescent ligand. Displacement of the fluorescent ligand
is essentially irreversible at a
large excess of unlabeled ligand. The fluorescence decrease follows a
single exponential decay with
the rate constant k–2. Very low rate constants are difficult to determine
directly because of the long
measurement times required, which requires a very stable signal to
follow the reaction.
48
10.2 You determine the observed rate constants for binding of an
ATPase to a non-hydrolyzable
ATP analog under pseudo-first-order conditions. The ATPase
concentration is 0.1 µM. kobs shows a
linear dependence on the concentration of the ATP analog in a
concentration range of 1 – 10 µM.
From the slope, you calculate k+ = 0.1 µM–1 s–1, the intercept is k– =
0.001 s–1. What are the possible
meanings of these rate constants?
Answer: From the linear dependence of kobs on the ligand
concentration, a binding model can be
inferred. In this case, k+ = k+1, k– = k–1, and Kd = k–/k+ = 1∙10–2 µM.
Alternatively, the linear dependence
may be the initial phase of the hyperbolic dependence for a two-step
binding mechanism, and
saturation (of the first equilibrium) has not been reached concentration
range studied. In this case,
we can approximate
𝑘𝑘𝑓𝑓𝑚𝑚𝑠𝑠 = 𝑘𝑘-2 +
𝐵
𝑘𝑘2
𝐵
𝐵𝐵0
0
≈ 𝑘𝑘-2 + 𝑘𝑘2
𝐵𝐵0 + 𝐾𝐾𝑢𝑢1 𝐾𝐾𝑢𝑢1
for B0 ≪ Kd1, and
𝑘𝑘+ = 𝑘𝑘2
𝐵𝐵0
𝐾𝐾𝑢𝑢1 ; 𝑘𝑘- = 𝑘𝑘-2
10.3 Binding of a substrate analog to an enzyme follows a two-step
mechanism with
k–
2 = 1∙10–5 s–1. You want to determine the overall Kd value in a
fluorescence equilibrium titration.
How do you perform the experiment?
Answer: Because of the small k–2, the overall equilibrium will only be
reached very slowly. The best
approach is to prepare the samples for each point of the titration
separately and let them equilibrate
overnight before measuring the signal. In a step-wise titration, you will
most likely miss the slow
phase in signal change and take a measurement before equilibration is
complete. The Kd value
determined form such a titration curve constitutes an upper limit for
the overall Kd.
49
Steady-state (enzyme) kinetics
11.1 The derivation of the Michaelis-Menten velocity equation is based
on the assumption that
[S] ≈ S0, which requires [S] ≪ KS. Derive the velocity equation for [S] ≈
KS.
Answer: The reaction velocity is v = kp[ES]. [ES] can be determined from
the solution of a quadratic
equation that describes the binding equilibrium as
[𝐸𝐸𝑆𝑆] =
𝐸𝐸0 + 𝑆𝑆0 + 𝐾𝐾𝑆𝑆
2 - ��𝐸𝐸0 + 𝑆𝑆20 + 𝐾𝐾𝑆𝑆�2 - 𝐸𝐸0𝑆𝑆0
Therefore,
𝑝𝑝 = 𝑘𝑘
𝑝𝑝 �𝐸𝐸0 + 𝑆𝑆20 + 𝐾𝐾𝑆𝑆 - ��𝐸𝐸0 + 𝑆𝑆20 + 𝐾𝐾𝑆𝑆�2 - 𝐸𝐸0𝑆𝑆0�
11.2 What is the velocity v relative to vmax at [S] = 2∙KS, 3∙KS, 5∙KS, and
9∙KS? What is the ratio of
substrate concentrations for which v = 0.9 vmax and v = 0.5∙vmax
([S]0.9/[S]0.5)?
Answer: For [S] = 2 KS, the reaction velocity is v = 2/3∙vmax, for [S] =
3∙KS it is v = 0.75∙vmax, for [S] = 5∙KS
we obtain v = 0.8∙vmax. The ratio of substrate concentrations that give
0.9∙vmax and 0.5∙vmax is
[S]0.9/[S]0.5 = 9∙KS/KS = 9.
50
11.3 Ethanol (EtOH) is oxidized to acetaldehyde by alcohol
dehydrogenase (ADH), using NAD+ as
an oxidant. Starting from a concentration of [EtOH] = 1.0 g L–1 in the
body, ADH reduces the ethanol
concentration by 10% in 1 h. ADH is saturated with EtOH throughout
the reaction, and NAD+ is
maintained constant. What is the reaction velocity in µM s–1? M(EtOH)
= 40.07 g mol–1. What are the
molar concentrations and the blood alcohol levels in ‰ at the
beginning and at the end, assuming
equal distribution throughout the body? Is this assumption valid? How
long does it take until the
blood alcohol level is below 0.3‰?
Answer: The concentration at the beginning is [EtOH] = 1.0 g L–1 =
1.0/40.07 M = 25.0 mM, which is
reduced to 0.90 g L–1 = 22.5 mM after 1 h. The blood alcohol level is
(approximately)
1 g L–1/1000 g L–1 = 1‰ and 0.9 g L–1/1000 g L–1 = 0.9‰. Ethanol is
membrane-permeable and will
distribute in the body.
The reaction scheme is
EtOH + NAD+ → CH3CHO + NADH + H+
At saturation of enzyme with substrate and constant [NAD+], the
reaction is a zero-order reaction
and
𝑝𝑝 = const. =
=
= 0.7 𝜇𝜇𝑀𝑀 𝑝𝑝-1
0.1 𝑚𝑚𝑚𝑚𝑚𝑚
0.1 𝑘𝑘
𝑚𝑚𝐻𝐻𝑟𝑟
40.07 𝑚𝑚 ∙ 3600
𝑚𝑚 ∙ 3600 𝑝𝑝
𝑝𝑝
At this velocity, the reduction of the blood alcohol level to < 0.3‰ takes
7 h.
11.4 The radical reaction of bromine and hydrogen to give HBr occurs
according to the following
reaction scheme:
Derive the rate law for HBr production under steady-state assumptions
for H∙ and Br∙ radicals. Why is
this assumption appropriate?