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Enzyme Kinetics

Fluorescence (%)

10

0 mM

1 mM

2 mM

3 mM

4 mM

5 mM

12.5 mM

8

6

4

2

0

0.001

0.01

0.1

Time (s)

10

100

Foreword

uninteresting. I have observed several explanations for

this:

The subject matter may be dicult to the

mathematically-challenged biology student.

Students may think that they will never undertake a

kinetic analysis and thus the study of kinetics is

unnecessary.

The student may find the subject matter accessible

but nevetheless uninteresting.

My goals in developing this guide to kinetics are:

reversible, occur on a time-scale of 10-12 to 109 sec

and are most frequently catalyzed by enzymes. The

study of reversible biological reactions, their timedependence and the mechanisms of enzymemediated catalysis is called enzyme kinetics.

Enzyme-kinetics is central to every biological process

that ever has or will be studied and is the basis for a

great many assays that are routinely undertaken in

every research laboratory. Understanding enzyme

kinetics is, therefore, important if we are to understand

biological processes and the limitations of the assays

we undertake in order to study these processes.

very powerful tool that biologists use to study

biological problems.

To show that the use of the tools is straightforward

when the underlying principles and assumptions

are appreciated.

To emphasize that the student does NOT have to

memorize equations and derivations - they are

included in this guide for your reference.

To provide examples of analyses.

To emphasize key points that student should

understand.

To provide formative, self-evaluation questions with

keys in order that the student can evaluate their

understanding of the concepts.

i

1.2

Equilibria

Fractional equilibration

C HAPTER 1

0.8

0.4

0.0

0.1

reactions?

100.0

1.2

Fractional Equilibration

spontaneously (by itself)?

10.0

Time (min)

chemical reactions. We ask:

1. What is a reversible chemical

reaction and what are its

characteristics?

1.0

0.8

0.4

0.0

0.1

1.0

10.0

100.0

Time (min)

graph) and human red cell ghosts (upper graph) at 4C.

S ECTION 1

Reversible Reactions

L EARNING OBJECTIVES

1. Reversible reactions never come to a halt they achieve an equilibrium in which the

forward and reverse reactions are

quantitatively balanced.

2. For any reversible reaction, there is a fixed

relationship between the conentration of

products formed and substrates remaining at

equilibrium.

A reversible reaction is one in which a

product can be formed from starting

material (substrate) and substrate can be

formed from the ending material

(product). This reaction never stops but

reaches an equilibrium in which the rate

of product formation from substrate is

identical to the rate of substrate

formation from product.

In a reversible reaction, there is a fixed

relationship between the concentrations

of reactants (substrate, S) and products

(P) at a given temperature in the

equilibrium mixture.

and reaction conditions.

3

hydrogen and iodine

H2 + I2

Glucose + ATP

k1

Glucose-6-phosphate + ADP

[HI]2

K eq =

[H 2 ][I 2 ]

k-1

K eq =

2HI;

=

[Glucose]e [ATP]e

k1

concentrations, k1 and k-1 are constants

describing the rates of forward and

reverse reactions respectively and Keq is

the equilibrium constant.

or, in general

pA + qB + rC

xD + yE + zF;

K eq =

[ A] p [B]q [C]r

The equilibrium constant for any specific

reaction (at a given temperature) is rather

like a fingerprint - it is unique.

equilibrium

If the concentration of any one of the

substances is altered in an equilibrium

mixture, the concentrations of the other

substances must change so as to keep

Keq constant.

In the reaction

k1

k -1

2. Define Keq in terms of [S]eq, [P]eq

K eq =

[Glucose]e [ATP]e

were at equilibrium and more S were

added?

equilibrium mixture by adding

exogenous ADP, [glucose-6-phosphate]

will fall by combination of glucose-6phosphate and ADP to increase

[Glucose] and [ATP]. The net eect, of

course, is that Keq is unchanged.

were at equilibrium and more P were

added?

S ECTION 2

reversible reaction

proceed?

following energy diagram:

Energy

intermediate X and the term

GA later.

Example 1

GA

S

G

P

Progress of Reaction

direction left to right. Why? Because the chemical

potential (energy) of S > P. (Note G is independent of

the path of the reaction).

Now consider Examples 2 and 3 below

Example 3

Example 2

determines the net direction of the reaction.

Energy

L EARNING O BJECTIVES

GA

P

S

G, is the chemical potential of the product(s)

minus the chemical potential of the reactant(s)

3. The reaction proceeds in the direction of high

chemical potential to low chemical potential

(G < 0)

GA

G

Energy

P

G = 0

Progress of Reaction

Progress of Reaction

potential of P > S). Reaction 3 above will proceed

equally in both directions (no net reaction) because the

chemical potential of S = P.

6

Forms of Energy

the ability of the molecule to perform work.

temperature and pressure.

through a change in temperature.

The chemical potential or partial molar free energy

of S, for example is given by:

S = S + RT ln[S]

free energy (G) and enthalpy (H) of a system (the

reactants) and changes in the entropies (S) of the

system plus the surroundings (universe) are related in

the following manner:

you can see that the chemical potential of S is directly

proportional to its concentration.

The free energy change (G) for the reaction S P is

given by:

G = P - S

At equilibrium, the rate of P formation is matched

exactly by the rate of S formation. Thus the abilities of

P to form S (to do work) and vice versa are identical.

Hence, at equilibrium,

P = S

Thus,

G = 0

G = H - TS

Thus:

G T

The irreversible increase in Entropy gives direction to

the reaction!

Imagine we have two glass flasks connected by a

valve. One flask has an internal volume of 10 mL and

the second has an internal volume of 100 mL. The 10

mL flask is filled with an ideal gas.

7

gas immediately expands to occupy both flasks. We

have all observed this type of behavior in one form or

another and know this to be a spontaneous reaction

( G < 0).

between gas molecules (H = 0). Thus:

In the reversible reaction

SP

1.

when the reaction is at equilibrium.

2.

when the reaction proceeds in a net direction from

left to right.

3.

and P when the reaction proceeds in a net

direction from right to left.

4.

H and TS?

G = -TS

The fall in free energy is due to increased S. The

molecules redistribute to maximize system entropy.

Summary

1. A reaction occurs spontaneously only if G is

negative (P < S).

2. A system is at equilibrium (forward and reverse

reactions are balanced) and no net change occurs

when G = 0 (P = S)

3. The forward reaction cannot occur spontaneously

when G is positive. An input of free energy is

required to drive the reaction.

4. G depends upon the free energy of the products

(final state) minus that of the reactants (initial state).

i.e. G is independent of the reaction mechanism.

5. The irreversible increase in entropy provides a

directional driving force for the reaction.

S ECTION 3

G and equilibria

understand 3 conditions.

The starting condition refers to the concentration of

reactants at the beginning of the reaction.

The equilibrium condition refers to the concentration

of reactants the reaction is at equilibrium.

The standard condition refers to the concentration of

reactants under standard conditions.

In the reaction:

A+BC+D

[C] [D]

G = G o + RTln [A] [B]

G is the standard free energy change

R = gas constant (1.98 cal/mol/degree)1

T = absolute temperature

LEARNING

O BJECTIVES

[A], [B], [C] and [D] are the molar activities of the

reactants under starting conditions.

equilibrium conditions.

2. The relationships between G, G and Keq.

3. Understanding that standard free energy

changes are additive and how this is exploited

in nature.

1one

temperature of 1 gram of water from 14.5 C to 15.5 C. One joule (J)

is that energy needed to apply a 1 newton force over a distance of 1

meter. 1 kcal (1000 cal) = 4.184 kJ (4184 J)

conditions

i.e.

Defining Keq as

[C] [D]

K eq = [A] [B]

G = -RT ln Keq

P = 1 Atmosphere

pH = 7.0

Rearranging gives us

T = 298K = 25C

Keq = e-G/RT

and [D] (and therefore G) under the conditions of the

reaction but is used only to describe G.

[A] [B]

remains constant

(i.e. the forward reaction is balanced by the reverse

reaction)

Thus the free energy equation becomes

[C] [D]

0 = G o + RTln [A] [B]

and

[C] [D]

G o =- RTln [A] [B]

=10-G/(2.303 RT)

Substututing R and T (25C) we obtain

Keq =10-G/1.36

when G has units of kcal/mol.

Thus an equilibrium constant of 10 corresponds to a

G of -1.36 kcal/mol (see Table below)

Go

Keq per M

1,000,000

10,000

100

10

1

0.1

0.01

0.0001

0.0000001

kcal/mol

-8.2

-5.5

-2.7

-1.4

0.0

1.4

2.7

5.5

9.5

kJ/mol

-34.2

-22.8

-11.4

-5.7

0.0

5.7

11.4

22.8

39.9

Note: H-bond energies range from 3 to 7 kcal/mol. van der Waals bond energies

are approximately 1 kcal/mol.

10

Thus

A B G1

A C Gtotal

[G6P]

K eq1 = [glucose] [P] = 3.9 x 10 -3 M -1

i

B C G2

Gtotal = G1 + G2

This principle of bioenergetics explains how an

endergonic reaction (Keq < 1) can be improved (more

product formed) by coupling it to a highly exergonic

reaction (Keq >>1) through a common intermediate.

Consider the synthesis of glucose6phosphate a

reaction that occurs in all cells:

Glucose + Pi

Glucose6phosphate + H2O

G = 13.8 kJ/mol

ATP + H2O

ADP + Pi

G = -30.5 kJ/mol

H20) and may be expressed as the sequential

reactions:

1)

Glucose + Pi

Glucose6phosphate + H2O

2)

ATP + H2O

ADP + Pi

sum:

ATP + glucose

K eq2 =

[ADP] [P]i

5

=

3.9

x

10

M

[ATP]

K eqtotal [glucose] [P] [ATP]

i

=Keq1 * Keq2 = 7.8 x 102

Thus by coupling ATP hydrolysis to G6P synthesis,

the Keq for G6P formation is raised by a factor of 2 x

105.

This strategy of coupling one reaction with a low Keq

(or G > 0) to a second with a high Keq (or G < 0) is

used by all living cells in the synthesis of metabolic

intermediates.

ADP + glucose6phosphate

11

Summary

1.

At equilibrium, G = 0

2.

and G < 0

3.

4.

5.

and G > 0

An endergonic reaction can be made more

favorable (i.e. Keq increases) by coupling it to a

second exergonic reaction via common chemical

intermediates. Here the G of the reactions are

summative.

At 25C, and equlibrium constant of 10

corresponds to a G of -1.36 kcal/mol

We will solve an exam-type question to illustrate

relationships between starting and equilibrium levels

of reactants and reaction spontaneity (direction of net

flux).

We use the isomerization of dihydroxyacetone

phosphate (DHAP) to glyceraldehyde 3-phosphate

(G3P) as our example.

Question:

Keq for the reaction DHAP G3P is 0.1 (log10 Keq

= -1.). Defining: G = G+ {1.36 x log10[G3P]/

[DHAP]} kcal/mol. When the starting

concentrations of DHAP and G3P are 0.2 mM and 2

M respectively (log10[G3P]/[DHAP] = -2).

F.

reaction cannot occur

spontaneously.

H. G = 2.72 kcal/mol and G = -1.36 kcal/mol

I.

reaction can occur spontaneously.

J.

Solution

A.

12

+1.36 kcal/mol.

C. When the initial concentrations of DHAP and G3P

are 2 x 10-4 M and 2 x 10-6 M respectively we can

substitute these concentrations and G into the

equation to obtain

D. G = 1.36 kcal/mol + (1.36 x -2) = -1.36 kcal/

mol

E.

can occur spontaneously when the species are

present at the concentration stated above.

Note however that no calculations were necessary.

Since Keq > starting [G3P]/[DHAP], the forward

reaction must occur spontaneously (more G3P must

be formed) since all reactions must proceed to

equilibrium. If starting [G3P]/[DHAP] had been > Keq

the reaction would have proceeded spontaneously

from right to left.

1. In biochemical reactions:

A. A reaction can occur spontaneously only when

the sum of the entropies of the system and its

surroundings < 0.

B. The most important criterion that determines

whether a reaction can occur spontaneously is

Go.

C. The change in energy of a system is independent

of the path of a reaction.

D. At chemical equilibrium where no net change in

[products] or [reactants] can occur, G > 0.

A reaction can occur spontaneously only if the

standard free energy change of the reaction is < 0.

2. When a reversible reaction between substrate

and product has achieved equilibrium:

A. The standard free energy change of the reaction is

always zero.

B. Subsequent addition of product will drive the

reaction to the right.

C. The chemical potentials of substrate and product

are equal.

D. The forward and reverse reactions halt.

E. Subsequent addition of an enzyme that

accelerates the reaction will drive the reaction to

the right.

13

-2). Defining: G = Go+ {1.36 x log10 [B]/[A]} kcal/

mol. When the starting concentrations of A and B

are 1 mM and 0.1 mM respectively (log10[B]/[A] = -1).

A. G = 1.36 kcal/mol and Go = -2.72 kcal/mol

B. Under the starting conditions stated above, the

reaction can occur spontaneously.

C. Go = 2.72 kcal/mol and G = -1.36 kcal/mol

D. The reaction will proceed from left to right.

E. The reaction will proceed from right to left.

14

S ECTION 4

catalysts

for a reaction to occur.

How can an enzyme accelerate a reaction without

shifting its equilibrium? To understand this we return

to the "Transition state theory" (Eyring, 1935).

Reactant molecules must overcome an energy barrier

and pass through an activated complex before

proceeding on to the product of the reaction.

The isomerization reaction

SP

is best represented by

SXP

where X is the activated complex or transition state.

In terms of an energy diagram

Example 1

L EARNING O BJECTIVES

Energy

doing so are neither chemically transformed at

the completion of the reaction nor do they

alter the equilibrium of the reaction.

GA

S

P

Progress of Reaction

pathways with lower energy barriers to

catalysis.

activation energy (GA) fall back to the ground state.

Those that achieve the transition state energy are

committed to form product.

15

any temperature T is given by

GA

S F = e - RT

pathways?

A number of mechanisms are observed:

SF = 9.9 x 10-9

SF = 0.158

increase in the fraction of S that can achieve the

transition state! In principle, the reaction would be

accelerated by 16,000,000-fold.

Enzyme thus provide alternative routes of reaction

which have lower energy barriers.

An enzyme could act in the following manner:

X

1. Covalent Catalysis

A nucleophile (electron-rich group with a strong

tendency to donate electrons to an electron-deficient

nucleus) on the enzyme displaces a leaving group on

the substrate. The enzyme-substrate bond is then

hydrolyzed to form product and free enzyme.

2. Acid-base Catalysis

e.g. Lysozyme cleaves the glycosidic bond between

C1 of N-acetylmuramic acid and C4 of Nacteylglucosamine of bacterial cell wall

polysaccharides. Glu35 of lysozyme donates a proton

to the oxygen of the polysaccharide glycosidic bond

thereby hydrolyzing the bond.

Energy

Energy

3. Proximity

X

S

X

X

S

P

Progress of Reaction

P

Progress of Reaction

state species but the ground states of substrate S and

product P are unchanged (G is unchanged). Thus the

reaction is accelerated and the equilibrium is

unchanged.

increase their proximity. Reaction rate is related to the

number of collisions of correct orientation. When an

enzyme binds its substrates it insures that their

orientation is precisely that required for reactivity.

4. Molecular Distortion

The enzyme active site undergoes a conformational

change upon binding substrate distorting the

substrate into a conformation resembling the transition

state species.

16

Summary

1.

1.

2.

from 10 kcal/mol to 0.5 kcal/mol accelerate a

reaction at 0C?

3.

energy barriers for catalysis.

4.

barrier and pass through an activated complex

before proceeding on to the product of the

reaction.

2.

energy barrier by introducing alternative reaction

pathways

3.

reactants and products therefore do not aect the

equilibrium of a reaction.

4.

reactions equally.

5.

through covalent catalysis, acid-base catalysis,

proximity eects or by molecular distortion or

combinations thereof.

17

S ECTION 5

Key to formative

evaluations

and S < P

4. At equlibrium, H = T S

Section 3

1. C (S increases; G not G; G=0 at equilibrium)

2. C (G=0, not G; more P produces more S; the

reaction continues; enzyme leaves Keq unaltered)

Section 1

1. Keq = k1/k-1

2. Keq = [P]eq/[S]eq

3. [S] would fall and [P] would increase so that Keq

remained unchanged.

4. [S] would rise and [P] would fall so that Keq

remained unchanged.

Section 2

1. At equilibrium, G =0 and S = P

2. When the reaction proceeds left to right, G < 0

and S > P

reaction must proceed right to left.

Section 4

1. Transition state theory states that reactant

molecules must overcome an energy barrier and

pass through an activated complex before

proceeding to product formation.

2. At 0 C & GA = 10kcal/mol, SF = e-10,000/(1.987*273) =

9.8 x 10-9. When GA falls to 0.5 kcal/mol, SF =

3. Covalent catalysis, acid-base catalysis, proximity

eects or by molecular distortion.

4. No! Keq is unchanged.

18

C HAPTER 2

Analysis of

time

dependent

processes

39

38

37

kobs

36

F

35

34

33

32

0

time in seconds

1. provide a set of tools to analyze

ad vestibulum volutpat. Nisl rhoncus

time-dependent processes

turpis est, vel elit, congue wisi enim

2. understand

the underlying

nunc

ultricies dolor

sit, magna tincidunt.

mechanisms

that

reaction

Maecenas

aliquam

estgovern

maecenas

ligula

rates

nostra.

Fraction reacted

This

chapter

time-dependent

Lorem

ipsumconsiders

dolor sit amet,

processes. We

seek

to:

suspendisse

nulla

pretium,

rhoncus

1.2

0.8

0.6

0.4

0.2

0

10

100

1000

time, msec

10

glossary

explain when this is true)

Your challenge will be to understand when dierent

names mean the same thing or dierent terms.

In general you will encounter 2 types of kinetic

constants in this and subsequent chapters.

Constants shown in lowercase (e.g. k1, k-1, kon, ko

etc) are typically rate constants with units of per

unit time (e.g. s-1) or per concentration per unit time

(e.g. M-1.s-1).

subsequent chapters. This reflects: 1) my poor editing;

2) the reality that multiple names for the same

constant are also found in the literature:

KS KD; KM Km KM(app); sometimes KM KM(app)

can be equal to KS KD (I will explain when this is

true)

kf kon; kr ko

Vmax Vm Vm(app)

Vmax etc) can have more complex meanings, have

units of concentration (M) or are rates (mol/sec) and

are related in some predictable way to multiple rate

constants

e.g. KM = (k-1+kp)/k1

You will also encounter a great many equations. You

do not need to memorize these (although you may do

so involuntarily particularly if you use them frequently).

My goal is for this pamphlet to serve as a useful

resource for you.

xx

Glossary

catalysis - the process by which an enzyme or

catalyst accelerates a reaction.

catalyst - an agent that accelerates a chemical

reaction but which is unchanged in amount or

chemistry at the end of the reaction.

chemical equilibrium - a reaction in which forward

and reverse reactions continue to proceed but are

quantitatively balanced

[Substrate]e i.e. the ratio of product formed : substrate

remaining at equilibrium

thermodynamics of reaction rates - enzymes

introduce alternative reaction pathways in which the

Gibbs free energy of activation is reduced

velocity or rates - amount of material (substrate,

product, # cells etc) consumed or produced or

number of events occurring per unit time

equilibrium - a state in which opposing forces are

balanced.

products - molecules produced by the action of

enzymes on substrates

reaction order - the dependence of the reaction rate

on [substrates]n when n is the number of substrates

which must interact to form a single molecule of

product.

substrates - molecules that are acted upon by

enzymes

xxi

S ECTION 1

Context

SFG since 1991 (8), but ultrafast probing of a

flash-heated SAM requires some elaboration. In

the SFG technique we used, a femtosecond infrared (IR) pulse at 3.3 mm with a bandwidth of

150 cm1 is incident on the SAM, coherently

exciting all the alkane CH-stretch transitions in

the 2850 to 3000 cm1 range, along with

electrons in the Au skin layer, producing an

oscillating polarization in both the Au and the

SAM layers. At the same time, a picosecondduration 800-nm pulse (visible) with a bandwidth of 7 cm1 is incident on the sample. The

visible pulse interacts with this oscillating polarization through coherent Raman scattering to

create a coherent output pulse at the IR + visible

frequency. This combined IR-Raman interaction

is forbidden (in the dipole approximation) in

centrosymmetric media because the secondorder susceptibility c(2) vanishes in such media.

Because the methylene CH2- groups of the

alkane SAM form a nearly centrosymmetric

solid, the SFG signal that we observed originated

predominantly from the Au surface and the

terminal methyl CH3 groups. The well-known

SFG spectrum obtained in ppp polarization (4),

moment (m/mIR)2, which is temperature dependent. (C) With an

instantaneous temperature jump to

1100 K, the methyl head groups

become orientationally disordered in less than 2 ps.

Some examples

W.Stuhmer et al.

A

of C8 (n = 7) and C18

(n = 17) SAMs without

heating pulses (blue) and

with flash-heating to

800C (red). (B) VRF for

a C8 monolayer. (C) VRF

for a C18 monolayer.

==

RCK1

1 SOpA

RCK3

J6pA

RCK4

4 0

RCK5

A~~~~~~4Op

,,,J@20pA

Fig.

20ms

chann

poten

G/Gm

788

10 AUGUST 2007

VOL 317

SCIENCE

www.sciencemag.org

1.2 T

Vibrational Response

Functions (VRFs) of SelfAssembled Monolayers. B

VRF for a C8 monolayer. C

VRF for a C18 monolayer

From: Wang et al.,

SCIENCE VOL 317, pp

787-790, 2007

however, are invariant and fall under the

general umbrella of kinetic analysis.

Normalized current

CTX-Biotin

1.0

0.5

0.0

Washout

200

-40

vol.8, pp.3235 - 3244,

Fig. 5. Conductance-voltage

relations1989.

of RCK channels. (A) Families

of outward currents in response to depolarizing

voltage steps. From

3

top to bottom RCKI, RCK3, RCK4, RCK5. The traces are responses

to 50 ms voltage steps from -50 to 40 mV in 10 mV intervals.

- Thiol Sugar Ensemble currents recorded

+Thiol

Sugar

from

macro-patches. Sampling at 10 kHz,

filtering at 3 kHz low pass. (B) Plots of normalized conductance

(G/Gm) versus test potential for different RCK channels (RCK1: open

circles; RCK3: crosses; RCK4: diamonds; RCK5: filled circles). To

obtain the conductance values the current at a particular test potential

was divided by the driving potential assuming a reversal potential of

- 100 mV. The lines showed the results of a non-linear least-squares

TCEP

fit of a Boltzmann isotherm (see Materials and methods) to the

conductance values. The maximal conductance (Gm) obtained by the fit

was used to normalize the data. The half-activation voltages in this

400

600 are -24

800mV (RCK1),

1000

1200

plot

-37

mV (RCK3), -30 mV (RCK4) and

-40 (s)

mV (RCK5).

Time

Sugar

- Thiol

Sugar of metabolically-labeled

control

Shaker

to voltage+ Thiol

from -60 to 0 mV are shown in Figure 7.

B Chemical

steps

channels with CTXBiotin.

Washout(A) CTXBiotin (10 nM) After TCEP

The

of

size

step

currents at 0 mV varied between

elementary

Initial

Initial

inhibits Shaker-IR K+ currents in CHO-K1 cells treated

0.46

and

1.02

pA

(RCK4)

pA

(RCK5). The single channel

with either 50 M thiol sugar 1 (h) or 0.5% ethanol as a

current - voltage

relations were measured in cell attached

vehicle (s). Only metabolically-labeled

(+thiol sugar)

patchesbywith

normal after

frog

Washout

channels were irreversibly blocked

CTXBiotin

aRinger's solution on the extracellular

side. For

all channels,

simple washout; inhibition was

completely

reversed

by an the current-voltage relation

50 ms

50 ms in

is linear

application

of 1 mM TCEP. Reaction

profiles

were range -20 to 20 mV. However, since

the voltage

monitored with a 200 ms, 40 this

mV is

pulse

every

15s. range for conductance estimation, we

a rather

narrow

measured

the

average

amplitudes at 0 mV membrane

- Thiol Sugar

+ Thiol Sugar

CFrom: Hua

Z, Lvov A, Morinpotential.

TJ, Kobertz

WR.

While

theChemical

RCK1, RCK3 and RCK5 channels have

I/I

I/I

control of metabolically-engineered

voltage-gated

K(+)

rather similar

1.0single-channel current amplitudes, that of the

1.0

channels. Bioorg Med Chem RCK4

Lett 2011.

channel is considerably lower (Table I).

1 nA

ranging from psec (10-12 s) to days (10 s) or

even longer

1 nA

L EARNING OBJECTIVES

needed to be chemically reversible. Although disulfide bond formation between CTX and thiol-containing sialic acids on the cell surface is an obvious chemoselective and cell friendly reaction, we

chose to label CTX with a bismaleimide that had an internal disulfide bond because maleimides are inherently more stable in water

than MTS reagents. Moreover, this subtle difference would allow

for delivery of a small molecule probe to the modified K+ channel

subunit after cleavage with reductant, which would be useful in

subsequent biochemical, biophysical or imaging experiments. To

simplify the synthesis of the bismaleimide, derivatization of CTX,

and ensure delivery of a molecular probe to a K+ channel subunit,

we set out to

5 synthesize a symmetrical bismaleimide (Scheme 1)

from cystamine dihydrochloride 2 that would allow for the facile

incorporation of a molecular probe in the final step of the synthesis. The amino groups of cystamine 2 were capped with 2 equiv of a

doubly amino-protected, activated ester of L-lysine 3. Selective

deprotection of the Fmoc protecting groups gave the symmetric

diamine 4. Addition of 2 equiv of the NHS-ester of 3-(maleimido)propionic acid and N-Boc deprotection afforded bismaleimide

5, which was subsequently biotinylated with 2 equiv of NHSBiotin to give biotin bismaleimide 6. CTX was then derivatized by

labeling a cysteine mutant of CTX (R19C)32 with 100-fold molar excess of biotin bismaleimide 6 to yield CTXBiotin, which was purified by reverse phase HPLC as we have previously described.17,19

With CTXBiotin in hand, we decided to change both the channel (ShakerIR)33 and the expression system (CHO-K1 cells) to

demonstrate that our approach was versatile and could be used

to label glycosylated ion-conducting subunits. Inactivation-removed Shaker is similar to Q1 in that it is an archetypical volt-

Conductance-voltage relations of

RCK channels. (A) Outward

currents in response to depolarizing

voltage steps. From top to bottom

RCKI, RCK3, RCK4, RCK5. The

traces are responses to 50 ms

voltage steps from -50 to 40 mV in

10 mV intervals.

-80

max

Initial

Washout

0.5

max

Initial

After

TCEP

Pharmacology

0.5 of RCK channels

22

A profile of the pharmacological sensitivity of the different

RCK channels to the K+ channel blockers 4-aminopyridine

chann

Hold

inact

low

depe

subs

at 2

Tabl

curr

RCK

sensi

inac

sensi

RCK

bloc

effec

Dis

Comp

RCK

An

funct

chan

ident

pa

prope

sing

shou

chan

func

memb

prel

struc

De

slowl

foun

inact

spina

chann

1988

whic

1. The time courses may be predictable from first

principles (e.g. an assumed reaction mechanism)

and thus an analysis yields important information

about mechanism.

Time (h)

Fig. I . Time course of 3-0-methylglucose uptake in isolated muscle fibres. Ordinate: ratio

of i n t r a ~ l l u l a ractivity t o extracellula~activity of 3-0-methy1glucose per equixra1ent

volume of bulk external solution, .4bscissa: time in hours. External sugar concentration,

Time course

of 3-0-methylglucose

uptake in isolated

muscle

of Balanus

1 m M Uptake

was measured using rc)nventional

and scintillator

probe (cells

0 )methods.

Kumber of points per conventional determination, five or more. The water content of

nubilis. Ordinate:

ratio of intracellular activity to extracellular activity of 3-0isolated fibres (70%) is shown by the continuous line above the points. The time a t

diame*r, 1352

p m ;k m pAbscissa:

rature.

half-equilibration

is shown by thedmhed

methylglucose

per equivalent

volumeline.

ofMean

bulkfibre

external

solution.

21 OC.

using conventional (filled squares) and scintillator probe (open circles)

The calculated rate of sugar uptake a t 30 min is 2 pmol .cm-*. s-I. Assuming

methods.

The water content of isolated fibres (70%) is shown by the

uptake is not saturated, the permeability of the barnacle muscle fibre, '2 (em. s-I),

continuous

line above the

points.

timeflux,

at half-equilibration

to 3-0-methy~g~ucose

is related

t o The

the sugar

J (mol. ernv2.s-I), byis shown by

the dashed line. Mean fiber diameter, 1352 m ; 21 C.

where So is the external sugar concentration (in01 . emp3).This corresponds to a value

From:

Carruthers, cm.

A. J.s-IPhysiol.

VOL 336, pp 377-396, 1983

which is some 3 4 orders of magnitude larger than the

of P of 2 x

of artificial

lipid bilayers to sugars (Jung & Snell, 1968; Lidgard & Jones,

Ribosomal R N A Stability permeability

and Growth

Conditions

19'75).

At equilibrium, the 3-0-methylglucose space of the fibre is 70 yo,which is in close

contentsof barnacle muscle (71 & 1 7; ; R = 5 ) .

sgreement with estimates of the xra%er

Assuming this water is not bound, these results shour that 3-0-methylglucose is not

accumulated by barnacle muscle and that the transfer of the sugar across the

sarco1emma is mediated by a passive, facilitated process.

EJat ofphloretia on mqzr uptuke. Re18tively low concentrations of phloretin inhibit

C""

Length of chose, h

tion

were made using an incubation period of 30 min in order to obtain accurate

measurements of t2heinitial rate of sugar uptake.

during

normal

and slow larval growth conditions. The perFirst-order

decay analysis

of Drosophila embryonic total RNA during

centage of embryonic RNA remaining a t various times duringa chase

normal

and

slow

larval

conditions.

The

percentage

of embryonic

with light yeast (normalgrowth

growth

conditions,

solid

circles) or dense

RNAalgae

remaining

at various

times open

during

a chase

normalThe

growth

(slow growth

conditions,

circles)

has under

been plotted.

values in(solid

solid circles

from

thegrowth

experiment

shown in (open

Fig. circles)

conditions

circles)areorderived

during

slow

conditions

1 as well

as regression

four other independent

experiments

(not shown).

is plotted.

The

lines indicate

that the

stability ofThe

embryonic

values in open circles are derived from the experiment shown in Fig.

RNA4 increases

from

48

h

115

h

if

the

larval

growth

rate

as well as one other independent experiment (not shown).

The is reduced.

regression lines indicate that the stability

of embryonic RNA increases from48h ( r 2 = 0.915) to 115 h ( r 2 = 0.892) if the larval

From:

Winkles

al., J. Biol. Chem.,Vol 269, pp 7716-7720, 1985

growth

rate iset

reduced.

be slightly enriched in rRNA, as would be expected if nonrRNA species decay faster than rRNA. Therefore, the halflife of total RNA isa good estimate of rRNA half-life.

To investigate whether the stability of embryonic rRNA

varied under differentlarval growth conditions, we then measured the half-life of this RNA using the L + D protocol as

described under "Materials and Methods." A representative

experiment using a dense chase is shown in Fig. 4. Adult

females were fed a yeast paste containing [3H]uridinefor 46

b.Some of the embryoslaid during the last 12 h of this

radioactive labeling period were transferred into a medium

containing 50% 13C,15N-labeled Chlorella cells, 50%

13C,2H,'5N-labeledChlorella cells, and [14C]uridine. First-in-

method of analysis may involve:

a. linearization of the data followed by linear

regression to obtain the constants related to

the underlying mechanism

b. nonlinear fitting to obtain the constants related

to the underlying mechanism

c. quality analysis to determine whether the

analysis is appropriate.

Goals

1. This chapter will review the classification of

reaction orders and the limitations of this

classification.

2. The tools available to analyze reactions and the

quality or appropriateness of the underlying

analysis.

measurements you may undertake in your own

research or observe when reading the work of other

researchers.

23

S ECTION 2

Reaction order

Rate of a reaction

The rate or velocity, v, of a reaction or process

describes how fast it occurs.

The velocity is expressed as a change in

concentration (C) per unit time (t),

dC

v = dt

but may also express the change in a population of

cells with time, the increase or decrease in the

pressure of gas with time or the change in absorption

of light by a colored solution with time.

The order of a reaction

describes how the velocity of the reaction depends

upon the concentration of reactants.

In the (irreversible) isomerization reaction

L EARNING O BJECTIVES

1. Reaction orders

1. Zero-order

2. First order

3. Second order

1. Class 1

2. Class 2 (pseudo first-order)

k1

d [B]

m

=

= k 1 [A]

k

1 [A]

dt

Since m = 1, this reaction is first order with respect to

A and since A is the only independent concentration

variable in the rate equation, the reaction is overall

first-order.

24

The units for this first order reaction are derived from

moles of product formed per second per mole of

reactant or,

M per sec = k 1 M

M per sec

= k 1 = per sec

M

experimentally

Understanding the stoichiometry of a reaction is not

sucient to predict the rate law of the reaction. This is

illustrated in the table below.

E+S

k1

E$S

d [ES]

1

1

=

k

1 [E] [S] = k 1 [E] [S]

dt

Because m = 1 for both species the reaction is firstorder with respect to E or S but is second order overall

as one single step is involved in the reaction of two

species.

The units of this second order reaction are derived

from moles of product formed per second per mole2 of

reactants or

molarity per sec = k1 (molarity)2

M per sec

= k 1 = per M per sec

M2

by the reaction, it is frequently omitted in the rate-law

expression. For example, with the first reaction, a

more complete rate law is:

v = k[sucrose][H+][H2O]

(the reaction is 3rd order overall). However, H+ is a

catalyst and its [ ] is constant during the run; [H2O]

(solvent) is little changed because of its vast excess

(55.5 M). Thus the terms [H+] and [H2O] are omitted in

the rate law. If the reaction were carried out at varying

[H+] or in an inert solvent, a first-order dependence of

the reaction on [H+] and on [H2O] is seen.

25

stoichiometry, NOT reaction mechanism or order.

Reaction 3 Dinitrogen pentoxide decomposition

N2O5

NO2 + NO3

NO3

NO2 + O

2O

O2

indicated to balance the equation. It could just as

easily have been written as: N2O5 2NO2 + O2

Reaction 4 Nitrogen dioxide decomposition to nitric

oxide and oxygen.

This involves formation of an intermediate thought to

be the one shown in the reaction below

To understand how we can distinguish reaction orders

experimentally, we will examine the following

reactions1

Zero-order kinetics

1The

This file is a GraphPad Prism file that contains the data for each type

of plot shown and the types of analysis made.

If you wish to plot these data yourself, you should download the file

from the Core Curriculum website:

2NO2

ONOONO

2NO + O2

http://inside.umassmed.edu/uploadedFiles/gsbs/courses/

2012-2013_Core_Course_Files/CoreKinetics.pzf%20-%20for

%20students%20only.zip

and download the GraphPad Prism version 6 software from

Prism 6 Winhttp://cdn.graphpad.com/downloads/prism/6/

InstallPrism6.exe

Prism 6 Windows serial number: GPW6-200512-LEM5-16772

Prism 6 Machttp://cdn.graphpad.com/downloads/prism/6/

InstallPrism6.dmg

Prism 6 Mac serial number: GPM6-200513-LEM5-F3EF2

26

NAD+).

A zero-order reaction

zero-order kinetics

Zero-order reaction

Substrate

Product

80

v (d[P]/dt)

[Substrate] or [Product]

10

100

60

40

20

100

200

[S] M

d [ethanol] d [acetaldehyde]

v ==

= k0

dt

dt

2

0

0

10

TIME

[product] increases linearly with time.

The negative sign is used with reactant - ethanol because its concentration decreases with time. The

concentration of its product, acetaldehyde, increases

with time.

C0

to acetaldehyde by the liver enzyme, alcohol

dehydrogenase (ALDH). The oxidizing agent is

nicotinamide adenine dinucleotide (NAD+) and the

reaction can be written:

ALDH

+

buered via metabolic reactions that restore it rapidly,

the rate of this reaction in the liver is zero-order over

CH3CHO

CH3CH2OH

0

27

dC

dt = k 0

The units of k0 are molarity per sec. This is a zeroorder reaction because there is no concentration term

in the right hand of the equation.

Defining C0 as the concentration at zero time and C as

the concentration at any other time, the integrated rate

law is:

C = C0 + k0 t

y = y-intercept + slope * x

This is the equation for a linear relation between the

independent (time) and dependent (concentration)

variables.

We can therefore subject the raw data to linear

regression analysis to obtain C0 (y-intercept) and k0

(the slope).

10

[Substrate] or [Product]

rate law

Zero-order reaction

Substrate

Product

8

6

4

2

0

0

10

TIME

y = x-intercept + slope * x

Best-t
values" "

"

Slope" "

"

"

Y-intercept
when
X=0.0"

X-intercept
when
Y=0.0"

Goodness
of
Fit" "

R
squared"

"

"

"

"

"

"

Substrate" "

-1
0
"

"

10
0"

"

10.00""

"

Product"

1
0

0 0""

0" "

"

1.000""

1.000

"

"

"

"

Units

mols/sec

mols

sec

1. Plot of St or Pt vs time produces a straight line with

slope = -k (for St) or k (for Pt)

2. k has units of mols produced or consumed per unit

time

3. Zero-order, enzyme catalyzed kinetics are typically

observed at saturating [S]

28

A first-order reaction

A first-order reaction corresponds to the dierential

rate-law:

1stOrder

dC

dt = k 1 C

[A] or [B]

4

3

concentration units in k1 so we do not need to know

absolute concentrations - only relative concentrations

are needed.

[Substrate]

[Product]

The reaction

1

0

0

k1

2

10

TIME

fashion with time and [product] increases in a

curvilinear manner with time. This observation

indicates that the reaction is NOT zero-order. How can

we analyze this further?

d [A] d [B]

v =- dt = dt = k 1 [A]

where k1 is the rate constant for this reaction.

The velocity may be expressed in terms of either the

rate of disappearance of reactant (-d[A]/dt) or the rate

of appearance of product (d[P]/dt).

29

Theory

- d [A] = k 1 [A] 0 t

[A] = [A] 0 e -k t

1

A at time zero and time t gives

ln [A] =- k 1 t + ln [A] 0

Half-life

Defining [A] at t1/2 as [A]0/2

slope x + intercept

ln2 0.693

t 1/2 = k = k

1

1

and because = 1/k1,

t1/2 = 0.693

30

Theory

all A is converted to B, [A] at any time t can be

calculated from [B] at time t as

[B] = [B] {1 - e -k t}

1

Thus we obtain

slope x + intercept

Half-life

Defining [B] at t1/2 as [B]/2

ln2 0.693

t 1/2 = k = k

1

1

and because = 1/k1,

t1/2 = 0.693

31

1stOrder

5

[A] or [B]

4

3

[Substrate]

with radioactive decay.

[Product]

A second clue comes from the measurement of halftimes. As [Substrate] declines from 5 - 2.5 mM, from

2.5 - 1.25 mM and from 1.25 to 0.625 mM, the time

required for each 50% reduction is unchanged at 1.4

sec.

1st Order

5

0

0

10

TIME

to an equilibrium value of 0 mM.

If we plot the log [substrate] vs time (or show the yaxis data on a log scale), we obtain

1stOrder

3

2

1.4 sec

1

0

0

10

1.4 sec

1.4 sec

10

TIME

[Substrate]

1

[A]

between log {[A]t - [A]} vs time indicate a first order

process. Let us now check this by applying a firstorder analysis to the data.

0.1

0.01

0

[Substrate]

10

TIME

order kinetics!

32

To do this we subject the data to nonlinear regression

(the plot is nonlinear) using an appropriate equation for

first-order reactions.

The integrated rate law for first-order substrate loss is

[A] = [A] 0 e -k t

1

parameters of the equation (k1 and [A]0) that generate

a curve that comes closest to the data. The result is

the best possible estimate of the values of those

parameters.

To use nonlinear regression, therefore, you must

choose a model or enter one. GraphPad Prism oers

a model for first-order reactions called One-Phase

Decay

The equation is:

Y=(Y0 - Plateau)*exp(-k*X) + Plateau

In which the parameters are defined as:

1.

2.

3.

4.

case because Y0-plateau = [A]0

steps:

1. Start with initial estimated values for each parameter in the

equation.

2. Generate the curve defined by the initial values. Calculate the

sum-of-squares - the sum of the squares of the vertical distances

of the points from the curve.

3. Adjust the parameters to make the curve come closer to the data

points - to reduce the sum-of-squares. There are several

algorithms for adjusting the parameters - Prism uses the

Marquardt algorithm.

4. Adjust the parameters again so that the curve comes even closer

to the points. Repeat.

5. Stop the calculations when the adjustments make virtually no

dierence in the sum-of-squares.

6. Report the best-fit results. The precise values you obtain will

depend in part on the initial values chosen in step 1 and the

stopping criteria of step 5. This means that repeat analyses of the

same data will not always give exactly the same results.

33

1stOrder

[Substrate] or [Product]

5

4

observed at subsaturating [S]

[Substrate]

[Product]

with slope = -k

starting value of St chosen.

2

1

0

0

with slope = -k

2

10

TIME

One phase decay"Perfect t"

Best-t values" "

Y0""

"

"

"

Plateau" "

"

"

k" "

"

"

"

Half Life""

"

"

Tau = 1/k"

"

"

Goodness of Fit"

"

Degrees of Freedom"

R square""

"

"

"

[Substrate]"

[Product]" "

Units

"

"

"

"

"

5.000""

0"

"

0.5000"

1.386
"

2.000""

"

"

"

"

"

0

5.000

0.5000

1.386

2.000

mM

mM

per
sec

sec

sec

"

"

48" "

1.000""

"

"

48

1.000

5. t1/2 = 0.693/k

6. k has units of time-1 (e.g. s-1). There are no

concentration units in k so we need not know

absolute concentrations - only relative

concentrations are needed.

7. k may be obtained by direct curve fitting

procedures using nonlinear regression

8. The full equation for loss of substrate is

[S]t = {[S]0 - [S]} e-(k.t) + [S]

9. The full equation for product formation is

[P]t = [P] (1 - e-(k.t))

10. When a first order reaction is reversible (as most

are), e.g.

k1

k2

k = k1 + k2

34

Second-order reactions

Fall into 2 categories in which the rate law depends

upon:

2. the product of the concentrations of two dierent

reagents.

Class 1 reactions (A+A P)

[Substrate]

[Product]

3

2

1

0

0

10

TIME

v=k[A]2

rate law for many reactions depends only on the

second power of a single component. e.g.

2 proflavin [proflavin]2

v = k [proflavin] 2

AAGCUU

2 AAGCUU

[A] or [B]

UUCGAA

v = k [A 2 GCU 2] 2

curvilinear fashion with time. This indicates that the

reaction is NOT zero-order. How can we analyze this

further?

The curves drawn through the points were computed

by nonlinear regression assuming first order kinetics

(one-phase decay equation). Note the systematic

deviations from the fit. This strongly suggests that this

reaction does not follow first order kinetics.

We can investigate this further by using GraphPad

Prism to plot the residuals of the fit (how each point

deviates from the calculated fit) vs time.

A non-random scatter of residuals around the origin

(perfect fit) would confirm a poor fit and that we

should consider either an error in data sampling or

another model for the data.

35

0.4

0.3

[Substrate]

[Product]

[A]0/[A]

0.2

of data

0.1

2nd order data

0.0

-0.1

-0.2

5

TIME

10

reaction

Theory of Class 1 Second-order Reactions

Defining [A] at zero-time = [A]0, it can be shown that

the integrated rate law is

1

1

[A] [A] 0 = k t

adding 1/[A]0 to both sides gives

1

1

k

t

=

+

[A] 0

[A]

0

0

10

TIME

circles, a second order reaction) as well as data from a

true first order reaction (closed circles) as suggested

by the 2nd-order linearized equation.

As you can see, transformation of the 2nd order data

produces a straight line with slope [A]0 k and yintercept = 1.

The slope [A]0 k indicates that the rate of loss of [A]

will increase linearly with [A]0

This is infact observed

[A] 0

[A] = [A] 0 k t + 1

Thus one expects a linear relation between the

reciprocal of the reactant concentration and time.

36

down when the reaction is reversible. Thus in the

reaction

15

1

2

3

4

5

6

7

8

9

10

[A]0/[A]

10

Increasing

[A]0

0

0

10

TIME

1.5

1.0

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

kr

when when kr kf/10

In fact, this general analysis of 2nd-order Class 1

kinetics derives from classical irreversible chemical

kinetics which have only limited application in biology.

2nd-order reactions - Class 2 (A+BP)

0.1320 2.842e-009

-3.974e-009 1.763e-008

3.010e-008

7.576

A reaction that is 2nd order overall is 1st order with

respect to each of the two reactants.

0.5

0.0

0

kf

A+A

2

E+S

10

[A]o

1. Standard 1st order analysis does not work

2. Plotting [A]0/[A] vs time produces a straight line

with slope [A]0 k

3. Plotting slope vs [A]0 produces a straight line with

slope k and y-intercept 0.

4. The units of k are concentration-1.time-1.

k1

k2

E$S

(e.g. [E] < [S]/100) and the substrate were varied, the

reaction dierential rate law is:

v=

d [ES]

dt = [E] k 1 [S] - k 2 [ES]

37

receptor (R).

R+L

kf

kr

R$L

as a log scale - this allows us to observe the data at

short time intervals more closely

Pseudo 1st Order

(note the forward and reverse rate constants have now been called kf

and kr but this name change is purely arbitrary - they could have been

called kon and ko or k1 and k2)

mixed with 1 nM R. The time course of LR formation

was monitored at each [L].

Pseudo 1st Order

[L]

0.0010

10

5.995

0.0008

3.594

[LR] M

2.154

0.0006

1.292

.774

0.0004

.464

.278

0.0002

.167

.1

0.0000

5

TIME

10

0.0010

10

5.995

0.0008

3.594

2.154

[LR] M

undergo a fluorescence change allowing measurement

of ligand binding. Alternatively, it may be possible to

measure ligand binding by use of radiolabeled ligand

and filter-bound receptor. Either way, the time course

of ligand binding may be examined to determine

whether it displays first or second order kinetics.

[L]

0.0006

1.292

.774

0.0004

.464

.278

0.0002

.167

.1

0.0000

0.1

10

TIME

GraphPad Prism and the one-phase association

equation. The fit is excellent in each case (the

residuals < [LR]/100)

You can also see that the reaction becomes faster at

higher [L] i.e. k increases and t1/2 falls with increasing

[L].

Each curve fit produces a value of k (typically called

kobs because it is an experimentally observed k) for

each starting [L].

We can analyze this further by plotting kobs vs [L]

38

kobs vs L

25

20

15

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

R+L

1.999 0.0001861

0.5012 0.0007352

-0.2507

0.5002

kr

R$L

d [LR]

v = dt = [R] k f [L] - k r [LR]

10

5

0

0

kf

6

[L] M

1. The slope is kf

2. The y-intercept is kr

3. The x-intercept is -kr/kf

10

shown that the integrated rate law is:

r

is e-t (kr + kf [L]) = e-t kobs.

2. Thus kobs = (kr+kf[L])

3. In a plot of kobs versus [L], kobs increases linearly

with [L] (slope = kf) and the y-intercept = kr.

4. The x-intercept (when kobs = 0) = -kr/kf. Why?

0=kr + kf[L]; thus -kf[L] = kr; thus -[L] = kr/kf

5. Analysis of the time course of L binding to R at

varying [L] permits computation of kf, kr and kf/kr =

Keq for the reaction.

6. This is ONLY true when [L] >> [R]. Here, first-order

kinetics are observed because [L] does not

change significantly. If [L] [R] the system will

behave like a class 1 second order reaction.

39

S ECTION 3

accurately by first order equations.

4. kobs for a class 2 2nd order reaction is kf[S]0 +

kr and when [S]0 is 0, kobs = kr.

Summary for Reaction order and kinetics

reaction and a second-order reaction that behaves

like a first order reaction?

1. A true first-order reaction is characterized by a

rate-constant, k, that is independent of

[substrate] or [product]. t1/2 is independent of

[substrate].

2. A second-order reaction that behaves like a

first order reaction (e.g. see this plot) is called a

pseudo-first-order reaction. Its rate constant,

kobs, increases linearly with [S] (i.e. kobs = kr

+kf[S]). t1/2 falls with increasing [substrate].

2. What is the dierence between a class 1 second

order reaction and a class 2 second-order

(pseudo-first-order) reaction?

1. A class 1 2nd order reaction is not described

accurately by first order equations but when 1/

[S] is plotted vs time, the plot is linear.

2. kobs for a class 1 2nd order reaction is k[S]0

and when [S]0 is 0, kobs = 0

the reaction depends upon the concentration of

reactants.

1. In the reaction, E+S ES, the reaction is first

order with respect to [E] at fixed [S], first order

with respect to [S] at fixed [E] but second-order

overall.

2. In the reaction, S+S P, the reaction is secondorder with respect to [S].

2. Zero-order reactions occur at a constant rate even

as substrate levels fall.

1. A plot of [S] vs time for a zero-order reaction is

linear with slope = -k

2. k has units of mol consumed or produced per

unit time.

3. First order reactions are non linear with time

1. A plot of St vs time is described by

[S]t = {[S]0 - [S]} e-(k.t) + [S]

2. Plot of Pt vs time is described by

[P]t = [P] (1 - e-(k.t))

40

3. t1/2 = 0.693/k

4. k has units of time-1 (e.g. s-1).

5. The half-time (t1/2) and k are invariant of the

starting value of [S].

4. There are two classes of second order reactions.

1. In reactions where two molecules of a

substrate combine to form a product (Class 1

reactions), the reaction is non linear with time

function of time, each curve is described by

first-order kinetics but now:

1. kobs increases and t1/2 decreases with [S]

2. kobs = kr + kf [S]

3. kf has units of M-1.s-1 and kr units of s-1

4. Keq can be obtained as kf / kr

= [S]0 k. This slope has units of per sec

2. Plots of [S]0 k vs [S]0 are linear with yintercept = 0 and slope = true k.

3. These rules break down for reversible

reactions.

4. k has units of concentration-1 time-1 (e.g.

M-1.s-1).

2. In reactions where two dierent molecular

species combine to form a product (Class 2

reactions), the reaction is non linear with time

1. If one species (e.g. an enzyme or receptor)

is held at a fixed and very low [ ] relative to

its substrate or ligand, the reaction is

pseudo-first order with respect to

[substrate] or [ligand].

41

S ECTION 4

Formative self-evaluation

questions

time never the best approach to confirm a 1st order

reaction? At fixed [enzyme], what concentration of

[S] produce zero-order kinetics?

11. At fixed [enzyme], what concentration of [S]

produce zero-order kinetics?

by answering the following questions

produce first-order kinetics?

1.

What are the units of zero-, first- and secondorder rate constants?

2 second-order kinetics?

2.

predict reaction order and mechanism?

behave like a first-order reaction?

3.

reaction?

Class 1 second-order reaction?

4.

rate constant?

second-order reaction vary with [S]?

5.

on time?

6.

18. How does kobs vary with [S] for a Class 2 secondorder reaction?

7.

with [S]?

and kr for a Class 2 second-order reaction?

8.

kinetics and Class 2 second-order kinetics?

9.

St vs time not always the best approach to

confirm a 1st order reaction?

pseudo-first order kinetics?

42

S ECTION 5

Key to formative

evaluations

= -k. Plotting [P] vs time yields a straight line with

slope = k.

5. No. Plots of [S] or [P] vs time are curvilinear.

6. t1/2 or the half-time of a reaction is the time

required for [S] to decrease by 50%.

7. t1/2 for a true first-order reaction is independent of

the starting [S].

8. t1/2 = 0.693/k

line if all of S is converted to P. What will work in all

cases is to plot log([S]t - [S]) vs time where [S] is

that concentration of S that remains when the

reaction achieves equilibrium.

2. first-order = per sec (per unit time)

3. second-order = per M per sec (per amount per

unit time)

2. No. The order of a reaction and mechanism must

be determined experimentally. Stoichiometry

simply shows a balanced reaction.

3. A zero-order reaction proceeds ([S] falls or [P]

increases) linearly with time.

straight line because [P] increases with time. You

have to invert the [P]t vs time data so that it now

resembles [S]t vs time data. This can be achieved

by measuring [P] (that concentration of P

produced when the reactions attains equilibrium)

then calculating [P]-[P]t and plotting that result vs

time. Thus a plot of log([P]-[P]t) vs time will

43

[P]0 (that [P] present at zero-time) is 0. If [P]0 > 0

then [P]0 must be subtracted from [P] and [P]t for

this analysis to work.

11. Zero-order kinetics are observed when [S] >>> Km.

12. First-order kinetics are observed when [S] <<< Km.

13. A Class 1 second-order reaction describes a

reaction in which 2 molecules of a single molecular

species combine to form a product e.g. S + S P.

A Class 2 second-order reaction describes a

reaction in which 2 molecules of dierent

molecular identities combine to form a product e.g.

E + S P.

linearly with [S] when the concentration of the

second reactant is held constant and is << [S].

19. A plot of kobs vs [S] produces a linear plot with

slope = kf, y-intercept = kr and x-intercept = -Kd

20. In a true first-order reaction, k and t1/2 are

independent of [S]. In a Class 2 second-order

reaction, kobs increases with [S] and t1/2 falls with

[S].

21. A Class 2 reaction therefore resembles a true firstorder reaction at any specific [S] but unlike a truefirst order reaction k and t1/2 vary with [S]. Hence

the moniker of pseudo.

when E is held at a low and constant level, the

reaction will show a first-order dependence on S

and vice-versa.

15. By plotting [S]0/[S] vs time. This produces a

straight line with slope [S]0 k.

16. k is independent of [S]0.

17. When the reaction is reversible.

44

C HAPTER 3

Steady-state

kinetics of

enzymecatalyzed

This chapter considers receptor-ligand

Lorem ipsum

sit amet,

equilibria

and dolor

enzyme

catalyzed

suspendisse

rhoncus

reactions. Wenulla

seekpretium,

to:

tempor placerat fermentum, enim integer

1. rationalize simple Michaelis-menten

ad vestibulum volutpat. Nisl rhoncus

behavior

turpis est, vel elit, congue wisi enim

nunc

ultricies

dolor

magna

tincidunt.

2. provide

a set

of sit,

tools

for routine

Maecenas

est maecenas ligula

analysisaliquam

of enzyme-catalyzed

nostra.

reactions

E

C

I

I

S

E

I

C

kp

E+P

Ki

EI

+

S

Ks

ES + I

C

kp

E+I

+

S

E+P

ESI

substrate S combine to form a complex.

Context

upon binding S.

2. The spectroscopic characteristics of E and S can

change upon ES formation.

3. High specificity for ES formation is observed.

4. The ES complex may be isolated in pure form.

5. At constant [E], increasing [S] results in increased

product formation to a point where product

formation no longer increases. This saturation is

presumed to reflect the fact that all E is now in the

form ES. This is illustrated below.

L EARNING OBJECTIVES

prior to the release of product (P) and regeneration of

free

These are:

1. E.Enzymes

are biological catalysts

1. ES complexes have been directly visualized by EM

2.and

Enzymes

combine reversibly with substrates

X-ray crystallography.

to form products

Jack Grith developed

techniques that let scientists

see the finer details of DNA. In

1971 he and Arthur Kornberg

published this photo, the first

electron microscope image of

DNA bound to a known protein

- DNA polymerase.

rationalized in terms of the Michaelis Menten

equation

120

enzyme (E)/substrate (S) complex, ES.

Vm

100

80

60 0.5 V

m

40

20

0

Km

0

10

20

30

40

50

[S] mM

xlvi

100

v (d[P]/dt)

75

50

25

0

0.01

0.1

10

100

1000

[S] [S]

mM

M

Km is that concentration of [S] producing a v of Vm/2.

rectangular hyperbola which in this instance takes the

generic form

Vm [S]

v = K + [S]

m

This equation is called the Michaelis-Menten equation.

Our challenge in this chapter is to understand why the

phenomenon of enzyme-mediated catalysis is well

approximated by this relationship.

xlvii

S ECTION 1

Michaelis-Menten Kinetics

into its various parts

2. Understanding enzyme-substrate interactions

1. Analyzing enzyme-substrate interactions

2. Introducing the catalytic step

3. Analyzing enzyme-catalyzed reactions

3. Inhibition of enzyme-catayzed reactions

1. Competitive

ES

kr

k2

E+P

k -p

complex ES. ES is then converted to EP which

dissociates to E and product (P). The rate constants

kf, kr, k2, k-2, kp and k-p describe the various steps

involved in the reaction:

kr, k2, k-2, and kp are first-order rate constants

kf and k-p are second-order rate constants

This may be represented as a reaction coordinate - an

abstract one-dimensional coordinate representing

progress along a reaction pathway.

(ES..EP)

(E...P)

EP

+

(E..S)

2. Noncompetitive

0

E+S

3. Uncompetitive

4. What does Km represent?

kp

EP

k -2

Energy

L EARNING O BJECTIVES

E+S

kf

ES

E+P

6. Kinetic perfection

Reaction coordinate

48

scheme is a very significant undertaking. Moreover,

the equation describing the rate of reaction in terms of

substrate and product levels and rate constants is

quite complex.

However, we can make a number of simplifying

assumptions in order to more readily obtain a solution

to this scheme. How do we do this?

negligible. While enzymes accelerate both forward

and reverse reactions to the same extent, we (as

the biochemists working with this enzyme) can

establish experimental conditions that preclude or

minimize the reverse reaction.

converts P into another species Q which cannot

react with our enzyme. Or, we can measure the

rate of reaction at very early time points where

the reverse reaction is insignificant.

now becomes:

E+S

kf

kr

ES

kp

E+P

1) Formation of ES

2) ES breakdown to product P and free enzyme E

The formation of ES is a second order process and the

breakdown of ES to E + S or to E + P are first order

processes.

The units are:

1.

kr = kp = per sec.

2.

units are important.

i.e. ES breaks down directly to E + P.

Make certain that [S] >> [E]. Thus the instantaneous

interaction of S and E to form ES does not

significantly aect free [S] (although [S] will slowly

fall due to its conversion to P).

49

interactions

kf

[ES]

k r [E] [S]

[E] t =

k

[E] + k f [E] [S]

r

the enzyme (E)/substrate (S) complex, ES.

Consider the reaction:

E+S

kf

kr

ES

kf

[ES]

k r [S]

[E] t =

k

1 + k f [S]

r

d [ES]

dt = k f [E] [S]

The rate of ES breakdown is given by:

complexed with S at any given [S], [E]t as

[ES]

[S]

=

[E] t

kr

k f + [S]

- d [ES]

dt = k r [ES]

Thus at equilibrium

d [ES] - d [ES]

dt = dt ` k f [E] [S] = k r [ES]

Hence

k

[ES] = k f [E] [S]

r

If we seek to understand how much substrate is

bound (i.e. [ES]) at any given [S] and [E], we can

express [ES] as a fraction of total enzyme [E]t as:

[ES]

[ES]

=

[E] t

[E] + [ES]

kr/kf has units of

per sec

per M per sec = M

When [S] = kr/kf, this means that

[ES]

[S]

[S]

=

=

[E] t

kr

2 [S] = 0.5

k f + [S]

This means that one-half of [Et] = [ES] when [S] = kr/kf

50

In the reaction

E+S

kf

kr

ES

constant for E and S interaction

kr/kf = 1/Keq = KS or Kd = the dissociation constant of

the ES complex.

When Kd is 1 M, 50% of Et = ES when [S] = 1 M

= 1 x 10-6M.

When Kd is 1 nM, 50% of Et = ES when [S] = 1 nM

= 1 x 10-9M.

A low value for Kd means that the ES complex is more

stable (less dissociates to E + S) thus at any [S], there

is a higher probability that ES is formed. Less S is

required to occupy one-half of the available binding

sites. The enzyme shows high anity for S.

energetically favorable interactions between E and S

(e.g. H-bonding and multiple van der Waals).

You may recall from Thermo that Keq = e-G/RT

Thus at 20C, an equilibrium constant of 10

corresponds to a G of -1.36 kcal/mol (see Table

below)

Go

Keq per M

1,000,000

10,000

100

10

1

0.1

0.01

0.0001

0.0000001

Kd M

0.000001

0.0001

0.01

0.1

1

10

100

10000

10000000

kcal/mol

-8.2

-5.5

-2.7

-1.4

0.0

1.4

2.7

5.5

9.5

kJ/mol

-34.2

-22.8

-11.4

-5.7

0.0

5.7

11.4

22.8

39.9

der Waals bond energies are approximately 1 kcal/

mol.

51

enzyme binding capacity?

In this experiment we continue our analysis of ligand

binding started on page 37. Here we measure the

amount of ligand bound to the receptor R at 10 sec

(where [L], [R] and [LR] are in equilibrium; see page

37).

equilibrium binding

[LR]
M

2.848e-004

3.995e-004

5.262e-004

6.496e-004

7.557e-004

8.377e-004

8.959e-004

9.349e-004

9.599e-004

9.756e-004

0.0010

0.0008

[LR] M

[L]
M"

0.100"

0.167"

0.278"

0.464"

0.774"

1.292"

2.154"

3.594"

5.995"

10.000"

are summarized on the next several pages and involve

linearization of nonlinear data.

Defining Bmax as

enzyme, we can rearrange the equation for ligand

binding to give the Michaelis-Menten equation for

ligand binding as

B max [S]

[S] b = K + [S]

d

0.0006

0.0004

the enzyme.

0.0002

0.0000

B max = [E] t n

10

[L] M

Prism by using the One-site binding - specific

binding equation

Bmax = 20 M

Y=Bmax*X/(Kd + X)

Where Bmax is the enzymes capacity to bind S

The result yields:

[LR] M

One site -- Specific binding

Best-fit values

Bmax

0.0010

Kd

0.25

M

M

52

Kd 1

1

1

=

+

[S] b B max [S] B max

obtain

[S]

Kd

Kd 1

1

1

=

+

=

+

[S] b B max [S] B max [S] B max [S] B max

This is a linear equation. Plotting 1/[S]b on the y-axis

and 1/[S] on the x axis, we obtain:

slope = Kd/Bmax,

y-intercept = 1/Bmax

Kd

1

1

1

1

=

+

=

+

[S] b B max K d B max B max B max = 0

[S]

[S]

K d [S]

Kd

1

[S]

=

+

=

+

B max

[S] b B max [S] B max B max

This is a linear equation. Thus plotting [S]/[S]b on the

y-axis and [S] on the x axis, we obtain:

slope = 1/Bmax,

y-intercept = Kd/Bmax

When [S] = -Kd

[S]

Kd

Kd

=

+

[S] b B max B max = 0

x-intercept = 1/-Kd

Lineweaver Burk

x-intercept = -Kd

1/[LR] per M

4000

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

251.0 0.09620

999.2 0.3800

-3.980

0.003983

[LR] M

10000

3000

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

999.9 0.03448

250.5 0.1362

-0.2505

8000

slope = Kd/Bmax

2000

y-cept = 1/Bmax

1000

[L]/[LR]

1/[LR] per M

x-cept = -1/Kd

Multiplying by [S]

6000

slope = 1/Bmax

y-cept = Kd/Bmax

x-cept = -Kd

4000

2000

-5

5

1/[L] per M

10

0

10

[L] M

53

3 ) Scatchard method

B max [S]

[S] b = K + [S]

d

[S] b K d + [S]

[S] b K d

B

=

max =

[S]

[S] + [S] b

Dividing by Kd gives

B max [S] b [S] b

K d = [S] + K d

Significance of Bmax

[S] b B max

1

[S]

=

b

Kd

Kd

[S]

This is a linear equation. Plotting [S]b/[S] vs [S]b yields:

slope = -1/Kd,

y-intercept = Bmax/Kd

When [S]b = Bmax

[S] b B max B max

[S] = K d - K d = 0

[LR] M

0.004

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

-3.984 0.002759

0.003986 2.095e-006

0.001000

-0.2510

0.003

0.0005

0.0010

If Bmax is measured as 10 M bound ligand or

substrate and you know that [E]t is 5 M,

0.001

0.000

0.0000

substrate and you know that [E]t is 10 M,

n = Bmax/[E]t = 1

slope = -1/Kd

y-cept =Bmax/Kd

x-cept = Bmax

0.002

concentration of bound ligand as a function of free

[ligand]), you typically measure the maximum binding

capacity (Bmax) of your system and Kd. Your challenge

is then to understand how Bmax is related to [Et].

B max = [E] t n

0.005

[ligand bound] or Bmax under the prevailing

experimental conditions.

x-intercept = Bmax

[LR]/[L]

non-linear regression analysis is the preferred method

used today. Why? Inverting data amplifies errors at

low [S] where experimental error (signal to noise) is

frequently greatest and todays computers are more

than fast enough to undertake the analysis.

0.0015

[LR] M

54

the enzyme

n = Bmax/[E]t = 2

substrate and you know that [E]t is 20 M,

n = Bmax/[E]t = 0.5

With some enzymes, the functional unit

comprises only 1 enzyme molecule which

binds 1 substrate or ligand. Here, [Et] = Bmax

and n = 1.

Other enzymes form a complex that

contains 2 or more functionally noninteracting enzyme molecules - each of

which binds 1 substrate or ligand.

Expressing [E]t in terms of [subunit]t, [Et] =

Bmax and n = 1

enzyme

Subunit

A

ES

S+E+I

Ki

EI

and I binding to the enzyme, E, respectively.

Ligand binding in the presence of a competitive antagonist

I

Subunit Subunit

A

B

1.0

[ligand]b/[Enzyme]t

Subunit B

Subunit A

0.8

2 or more functionally interacting, but

otherwise identical enzyme molecules. Here,

two enzyme molecules may be required to

form a single binding site, and n = 0.5.

Kd

[I]=0

[I]=5

[I]=25

[I]=50

[I]=100

0.6

to the right (Kd

increases) but the

maximum amount of

binding (Bmax) is

unchanged.

0.4

0.2

0.0

10

100

[S] M

1000

10000

55

described by:

[S] b =

B max [S]

[I]

K d (1 + K ) + [S]

i

constant for I binding to E.

Introducing catalysis

Let us now consider the breakdown of ES to

regenerate free enzyme E and release the product, P.

The overall scheme is visualized as

E+S

[I]

K dapp

Kd - 1 = Ki

[I]

Ki = K

dapp

Kd - 1

Thus computation of Kd in the absence of I and Kdapp

at any given [I] permits computation of Ki.

kr

ES

kp

E+P

presence of inhibitor

[I]

K dapp = K d (1 + K )

i

kf

v = kp [ES]

We must again express [ES] in terms of known

quantities

Rate of [ES] formation

+d[ES]/dt = kf [E][S]

Rate of breakdown of [ES]

-d[ES]/dt = kr[ES] + kp [ES]

or

-d[ES]/dt = (kr + kp) [ES]

In the "steady state" the concentrations of

intermediates (e.g. ES) are unchanged, whereas [S] +

[P] can change. If we limit measurements of v to early

stages, [ES] does not change (there is no reverse

reaction)

d[ES]/dt = 0

56

[S]

v

=

kr + kp

k p [E] t

k f + [S]

thus

kf [E] [S] = (kr + kp) [ES]

hence

[E] [S] k r

[E] [S]

[ES] = k + k = k + k

r

p

r

p

kf

The following steps are algebraic tricks:

[E]t = [E] + [ES]

dividing the velocity equation by [E]t we obtain

k p [ES]

v

=

[E] t [E] + [ES]

divide both sides by kp

and defining:

Vm = kp [E]t

and

Km =

kr + kp

kf

Vm [S]

v = K + [S]

m

[ES]

v

=

k p [E] t [E] + [ES]

substituting for [ES]

kf

k r + k p [E] [S]

v

k p [E] t =

k

[E] + k +f k [E] [S]

r

p

canceling [E]

kf

k r + k p [S]

v

k p [E] t =

k

1 + k +f k [S]

r

p

57

At very high [S],

vV

zero-order

kinetics

increasing [S]

100

product formation in an enzyme catalyzed reaction

and express this rate, v, versus [S].

80

Enzyme

Vm [S]

v. K

m

i.e. v increases linearly with [S]

60

rate of reaction M/min

at low [S]

v (d[P]/dt)

80

40

20

0

first-order

kinetics

0

100

60

40

M/min

100.0

25.00

Vm

Km

20

200

[S] M

many instances it is not possible to add sufficient

quantities of substrate to saturate the enzyme.

You may then ask, if Vm is not measurable, how

does one determine Vm and Km for a reaction?

The very same tricks that helped us with ligand

binding also help us here.

Why? Because the Michaelis Menten equation

and the saturable ligand binding equation take

the same form:

0

0

20

40

60

[S] M

Michaelis-Menten equation yields Vm and Km.

There are other methods of calculating these

parameters by linearization of the data and by using

linear regression.

You should note, however, that linearization greatly

amplifies errors of analysis!

Const $ x

y = Const 1+ x

2

58

V m [S]

v = K + [S]

m

Hanes-Wolf Analysis

The Lineweaver Burk equation is

[S]

Km

1

=

+

v

V m [S] V m [S]

1 Km 1

1

=

+

v

V m [S] V m

Multiplying both sides by [S] gives

thus

1 Km 1

1

=

+

v

V m [S] V m

plotting 1/v vs 1/[S] yields a straight line with slope =

Km/Vm and y-intercept = 1/Vm

0.15

1/v min/ M

0.2500 1.824e-009

0.0100 2.711e-010

-0.0400

4.000

[S] K m

1

=

+

[S]

v

Vm

Vm

Vm and y-intercept = Km/Vm

Hanes Wolf Enzyme

1.0

0.10

0.8

slope = Km/Vm

0.05

x-cept = -1/Km

y-cept = 1/Vm

-0.1

Thus

0.0

0.1

0.2

1/[S] per M

0.3

0.4

-1

Km

1

1

1

=

+

=

+

=0

v

Vm K m Vm Vm Vm

Thus the X-intercept = 1/-Km

1/v min/ M

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

v = V m [S] + V m

0.6

slope = 1/Vm

y-cept = Km/Vm

x-cept = -Km

0.4

0.2

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

1/slope

-20

-0.2

20

[S]

[S]/v

0.0100 2.320e-010

0.2500 7.331e-009

-25.00

100.0

40

60

[S] K m - K m

v = Vm + Vm = 0

Thus the x-intercept = -Km

59

Enzyme Inhibition

Noncompetitive Inhibition

synthesized as drugs.

Competitive Inhibition

Schematic

C

I

King-Altman Diagram

E+I

+

S

EI

Ki

E

I

Ki

EI

+

S

Ks

ES + I

C

kp

E+I

+

S

ESI

C

kp

Ks

enzyme

E+P

ES

kp

E+P

I is not transformed into product. The inhibitor, I,

resembles the substrate, S. I reduces the rate of

reaction + S by reducing the proportion of enzyme in

the form ES

E+P

catalytically inactive. When S binds, the enzyme

undergoes a conformational change which aligns the

catalytic center, C, with the susceptible bonds of S; I

interferes with the conformational change, but has no

eect on S binding.

60

feedback inhibition.

Uncompetitive Inhibition

threonine in bacteria involves 4 steps mediated by 4

dierent enzymes.

E

+

S

C

+S

Threonine

threonine

Isoleucine

+I

KI

ES + I

C

I

kp

ESI

C

kp

deaminase

(TD). This enzyme is noncompetitively inhibited by

isoleucine.

Thus as product increases, the first step in the

reaction decreases. As P falls due to shut down of

TD, so TD is released from inhibition due to

dissociation of P from the TD.P complex and the

reaction cycle proceeds again.

E+P

E+P

conformational change which aligns the catalytic

center, C, with the susceptible bonds of S and which

also exposes an inhibitor binding site.

Thus I can only bind to the ES complex to form ESI

which is catalytically inactive.

20% of enzymes show uncompetitive substrate

inhibition which is now recognized as an important

regulatory mechanism - Bioessays 32: 422429, 2010 Reed,

Lieb & Nijout

61

equation takes the form:

V m (app) [S]

v= K

m (app) + [S]

Where:

Competitive Inhibition

V m (app) = V m

[I]

K m (app) = K m T 1 + K Y

i

should overcome inhibition caused at any [I] but it will

take more S to produce the equivalent v observed in

the absence of I. Thus Km(app) increases with [I].

Noncompetitive Inhibition

Vm

V m (app) =

[I]

T1 +

Y

Ki

K m (app) = K m

The law of mass action tells us that binding of S is

unaected at all [I] so Km(app) is unchanged by [I] but

since [I] reduces the amount of catalytic E, Vm(app) falls

with [I].

Uncompetitive Inhibition

Vm

[I]

T1 +

Y

Ki

Km

K m (app) =

[I]

T1 +

Y

Ki

V m (app) =

amount of catalytic ES, thus Vm(app) falls with [I]. Since

S increases the amount of E accessible to I, [ESI]

increases with [S] so it takes less [I] to saturate ES as

[S] increases. Thus Km(app) falls with [I].In the following

examples, we simulate an enzyme E with Km and

Vmax of 25 M and 100 mol/min respectively.

We then inhibit the enzyme using 3 separate inhibitors

Competitive - Ki = 10 M

Noncompetitive - Ki = 10 M

Uncompetitive - Ki = 10 M

Control and inhibited rates of catalysis were measured

at 2.5 - 100 M substrate in the absence (control) or

presence (inhibited) of 10 M inhibitor.

62

Noncompetitive Inhibition

Competitive inhibition

100

100

inhibited

v mol/min

v mol/min

80

Control

80

60

40

Control

Michaelis-Menten Perfect fit

Best-fit values

Vmax

100.0

Km

25.00

20

0

0

Control Inhibited

Michaelis-Menten Perfect fit

Best-fit values

Vmax

100.0

50.00

Km

25.00

25.00

60

40

20

inhibited

Perfect fit

0

0

100.0

50.00

50

[S] M

Control

Inhibited

50

[S] M

100

100

Hanes Wolf Noncompetitive

control

Inhibited

Best-fit values

Slope

0.0100 2.100e-010 0.0100 2.572e-010

Y-intercept when X=0.0 0.2500 1.047e-008 0.5000 1.282e-008

X-intercept when Y=0.0 -25.00

-50.00

[S]/v

M.min/mol

control

Inhibited

1.5

[S]/v

M.min/mol

1.0

control

Inhibited

0.5

-25

-50

control

Inhibited

Best-fit values

Slope

0.0100 2.100e-010 0.0200 4.200e-010

Y-intercept when X=0.0 0.2500 1.047e-008 0.5000 2.093e-008

X-intercept when Y=0.0 -25.00

-25.00

50

25

50

75

100

[S] M

100

[S] M

Lineweaver Burk Noncompetitive

0.25

control

inhibited

Best-fit values

Slope

0.2500 1.371e-009 0.5000 3.877e-009

Y-intercept when X=0.0 0.0100 2.134e-010 0.0100 6.035e-010

X-intercept when Y=0.0 -0.0400

-0.0200

0.25

0.20

control

inhibited

0.15

1/v

min/mol

1/v

min/mol

0.20

0.10

0.05

0.0

control

inhibited

Best-fit values

Slope

0.2500 1.371e-009 0.5000 3.877e-009

Y-intercept when X=0.0 0.0100 2.134e-010 0.0200 6.035e-010

X-intercept when Y=0.0 -0.0400

-0.0400

control

inhibited

0.15

0.10

0.05

0.1

0.2

1/[S] per M

0.3

0.4

0.0

0.2

1/[S] per M

0.4

63

Uncompetitive Inhibition

Control Inhibited

Michaelis-Menten Perfect fit Perfect fit

Best-fit values

Vmax

100.0

50.00

Km

25.00

12.50

100

v mol/min

80

Km(app)

Vm(app)

Ki

mol/min

None

25

100

Competitive

50

100

10

Noncompetitive

25

50

10

Uncompetitive

12.5

50

10

Inhibition

Control

Inhibited

60

40

20

0

0

50

[S] M

100

[S]/v

M.min/mol

-25

control

Inhibited

Best-fit values

Slope

0.0100 2.100e-010 0.0200 2.749e-010

Y-intercept when X=0.0 0.2500 1.047e-008 0.2500 1.370e-008

X-intercept when Y=0.0 -25.00

-12.50

control

Inhibited

25

50

75

100

[S] M

1/v min/M

Best-fit values

Slope

Y-intercept when X=0.0

X-intercept when Y=0.0

0.10

control

inhibited

0.2500 1.371e-009

0.0100 2.134e-010

-0.0400

0.2500 3.358e-009

0.0200 5.227e-010

-0.08000

Vm

V m (app) - 1

determine Ki experimentally for Competitive and

noncompetitive inhibitions.

control

inhibited

plotting 1/v vs [I] yields a Dixon plot

0.05

0.0

[I]

[I]

Ki = K

m (app)

Km - 1

-0.1

Ki is calculated as

Ki =

0.15

Determining Ki

0.1

0.2

1/[S] per M

0.3

0.4

64

V m [S]

v= K

m (app) + [S]

and uncompetitive inhibitions

0.5

thus

Noncompetitive

[I]

T

Y

+

1

K

m

[S]

Ki

1 K m(app)

1

=

+

=

+

v

Vm

V m [S]

V m [S]

V m [S]

0.4

1/v min/M

Competitive

Uncompetitive

0.3

hence

Km

1 T Km

1

Y

=

+

[I]

v

K i V m [S] V m [S] + 1

0.2

0.1

-60

-40

-20

K

slope = K V m[S]

i m

20

40

60

[I] M

1 Km

y - intercept = V T [S]

+ 1Y

m

when 1/v = 0

Km

Km

[I] K [S] = T [S]

+ 1Y

i

-Ki(1+[S]/Km)

The x-intercept for non competitive inhibition yields

and

[S] K m

[S]

[I] = - K i K T [S]

+ 1 Y = -Ki T 1 + K Y

m

m

-Ki.

-Ki(1+Km/[S])

65

V m (app) [S]

v = K + [S]

m

thus

thus

[I]

[I]

T1 +

Y T1 +

Y

K

m

[S]

Ki

Ki

Km

1

+ V

v = V m(app) [S] + V m(app) [S] =

V m [S]

m

hence

and

[I] 1 K m

1 T

Y

T

Y

=

+

1

v

K i V m [S] + 1

1

1 1 T Km

1 T Km

Y

Y

=

+

+

[I]

1

v

K i V m [S]

V m [S] + 1

1 1 Km

slope = K V T [S]

+ 1Y

i m

1 Km

y - intercept = V T [S]

+ 1Y

m

when 1/v = 0

- 1

Km

1 1 Km

[I] K V T [S]

+ 1 Y = V T [S]

+ 1Y

i m

m

and

V m(app) [S]

v= K

m(app) + [S]

1 Km

1 Km

[I] V T [S]

+ 1 Y = - K i V T [S]

+ 1Y

m

m

thus

[I] = - K i

K m(app)

[S]

Km

1

=

+

=

v V m(app) [S] V m(app) [S] V m [S] +

hence

T1 +

[I]

Y

Ki

Vm

1

1 T Km

1

Y

=

+

+

1

[I]

v V m [S]

K i Vm

1

slope = K V

i m

1 Km

y - intercept = V T [S]

+ 1Y

m

when 1/v = 0

- 1

Km

1

[I] K V = V T [S]

+ 1Y

i m

m

and

[I] - K m

T

Y

K i = [S] + 1

Km

x - cept = [I] = - K i T [S]

+ 1Y

and

Ki =

[I]

T Km + 1 Y

[S]

66

Naturally occurring (source and targets)

Naturally occurring (source and targets)

glucose transporters)

Atropine (deadly nightshade; Acetylcholine

receptor)

Synthetic (their targets and use)

Ibuprofen (cyclo-oxygenase inhibitor - antiinflammatory)

Sulfanilamide (dihydropteroate synthetase;

antibacterials - inhibit folate synthesis)

Neostigmine (acetylcholinesterase; prolong

neuromuscular transmission - treat myasthenia

gravis)

Haloperidol (brain & endothelial cell Nitric Oxide

Synthase inhibitor - anti-psychotic)

Trichostatin A (Histone DeAcetylase; anti-cancer)

mycophenolic acid (Inosine monophosphate

transferase; Dengue virus)

see http://www.emdbiosciences.com/docs/docs/

LIT/ISB_USD.pdf

transmission and full-blown AIDS)

see http://www.emdbiosciences.com/docs/docs/

LIT/ISB_USD.pdf

67

Km is the concentration of [S] at which 1/2 of the

active sites are filled with substrate. The fraction of

filled sites [ES]/[E] t

Thus, when [S] = Km

[ES]

[S]

Km

=

=

[E] t

K m + [S] 2K m = 0.5

Hence one-half of the enzyme exists as ES and the

rate of the reaction v, is

of an enzyme if [Et] is known because

Vm = kcat [E]t

if [Et] = 1 M and Vm = 600 mmols/L/sec,

kcat = 6 x 105 sec-1 - Each round of catalysis is 1/kcat =

1.7 sec.

The expression Vm = kcat [E]t applies to all enzymes.

For the simple enzyme we are considering, the

solution for kcat is

kcat = kp

We also saw above that

Km =

kr + kp

kf

complexes, the algebraic solution for kcat is more

complex.

For example, in the reaction

k

Km = kr = Kd

f

where Kd is the dissociation constant for the ES

complex.

while in other enzyme-mediated reactions where kp

kr, Km > Kd.

E+S

ks

k-s

ES

k1

EP

k2

kp

E+P

k-p

k 1 k -p

k cat = k + k + k

-p

2

1

However, for the purposes of the discussion that

follows we shall assume that kcat = kp

68

When [S] << Km,

rate constant, ke can be calculated as

V m [S]

v. K

m

ke =

where

Vm = kcat [E]t

Since for our enzyme kcat = kp

this means that

kp

v = K [S] [E] t

m

v is therefore directly proportional to kp/Km and [S] at

fixed [E]t.

Are there limits upon kcat/Km?

kp

kp kf

=

Km kp + kr

Examination of this equation indicates that kf is

limiting. When kr << kp (i.e. catalysis is much faster

than ES dissociation into E and S),

kp kf

kp kf

.

kp + kr

kp = kf

Thus the greatest value that kp/Km can attain is kf. kf is

the second order rate constant that describes

association of E and S to form the ES complex. kf

includes terms that describe the collisional frequency

of E and S.

-1

M

.sec

1000

kT

D A = 6rr h

A

radii respectively of molecules A and B and N is

Avagadros number.

For an enzyme molecule of radius = 30 and a

substrate molecule of radius = 5 , the encounter rate

constant is 109 M-1.sec-1.

Thus diusion limits the rate of encounter of E and S

and an upper limit of kp/Km is 109 M-1.sec-1. Even this

requires that the substrate can encounter the enzyme

surface in any orientation.

kcat/Km ratios of some enzymes, e.g.

acetylcholinesterase and carbonic anhydrase are

between 108 - 109 M-1 sec-1 indicating they have

achieved kinetic perfection.

For these enzymes, because kp/Km 108 M-1 sec-1,

this means that kp is so fast relative to kr that the ES

complex breaks down faster to form product and E

than it dissociates back to S and E.

Their activity is limited only by the rate at which they

encounter substrate in solution. Any further gain can

69

can be achieved by sequestering substrates and

products in the confined volume of a multi-enzyme

complex, e.g. mitochondria.

For an interesting discussion of kinetic perfection,

read

Biochemistry 1988, 27, 1158-1167

Triosephosphate Isomerase Catalysis Is Diusion

Controlled.

Stephen C. Blacklow, Ronald T. Raines T. Wendell

A. Lim, Philip D. Zamore, and Jeremy R. Knowles

70

S ECTION 2

B max [S]

[S] b = K + [S]

d

4. What is the relationship between [E]t and Bmax?

B max = [E] t n

1. What assumptions did we make in order to derive

the Michaelis-menten equation for an enzyme

catalyzed reaction?

1. The reverse reaction (P S) is negligible.

2. Assume only a single central complex (ES)

exists. i.e. ES breaks down directly to E + P.

3. [S] >> [E]. The instantaneous interaction of S

and E to form ES does not significantly aect

free [S].

Ligand Binding

1. For the reaction

E+S

kf

kr

ES

sites per enzyme

5. How can we compute Kd and Bmax from

measurements of [S]b at varying [S]?

1. Nonlinear regression analysis of plots of [S]b vs

[S] using the Michaelis-menten equation to

obtain best estimates of Kd and Bmax.

2. Linear transformation of the [S]b vs [S] data:

1. Lineweaver-Burk (1/[S]b vs 1/[S]) gives

1. Kd = -1/x-intercept

2. Bmax = 1/y-intercept

2. Hanes-Wolf ([S]/[S]b vs [S]) gives

1. Kd = -x-intercept

2. Bmax = 1/slope

3. Scatchard ([S]b/[S] vs [S]b) gives

1. Kd = -1/slope

4. Kd = 1/Keq

2. Bmax = x-intercept

and [substrate] bound to an enzyme?

plots?

71

unchanged but Kd increases.

7. What is Ki for a competitive inhibitor?

1. Ki is the Kd for inhibitor binding to E - We call it

Ki in order not to confuse it with Kd for S

binding to E.

8. How do we compute Ki for a competitive inhibitor?

1. We obtain the dissociation constant for S

binding to E in the absence of inhibitor (true Kd)

and in the presence of inhibitor (measured as

Kdapp).

2.Ki is obtained as:

[I]

Ki = K

dapp

Kd - 1

Catalysis

1. During the period in which we measure a

reaction, the concentration of intermediates

(e.g. ES) do not change although [S] and [P]

may change somewhat.

10. What is the Michaelis-menten equation for

catalysis?

Vm [S]

v = K + [S]

m

11. How is Vm related to [E]t?

1. Vm = [E]t kcat

12. What is Km?

1. Km is that [S] where v = Vm/2

13. How is Km related to the rate constants describing

the reaction?

1. The answer is dierent for each specific kinetic

model.

2. For our reaction, Km = (kp+kr)/kf

14. How can we compute Km and Vm from

measurements of v at varying [S]?

1. Nonlinear regression analysis of plots of v vs [S]

using the Michaelis-menten equation to obtain

best estimates of Km and Vm.

2. Linear transformation of the v vs [S] data:

1.Lineweaver-Burk (1/v vs 1/[S]) gives

1. Km= -1/x-intercept

2. Vm = 1/y-intercept

3. Hanes-Wolf ([S]/v vs [S]) gives

1. Km = -x-intercept

2. Vm = 1/slope

15. How do inhibitors aect substrate/velocity plots?

1. Competitive inhibitors

1. Leave Vm unchanged

2. Increase Kmapp

72

2. Noncompetitive inhibitors

1. Decrease Vmapp

2. Leave Km unchanged

3. Uncompetitive inhibitors

1. Decrease Vmapp

as

x - cept = - K i

3. For uncompetitive inhibition, Ki is calculated

from

2. Decrease Kmapp

16. How do we compute Ki for an inhibitor?

1. By measuring Km and Vm for a reaction in the

absence of an inhibitor and Kmapp and Vmapp in

the presence of the inhibitor.

1. For competitive inhibition, Ki is calculated as

[I]

Ki = K

m (app)

Km - 1

2. For noncompetitive and uncompetitive

inhibition, Ki is calculated as

Ki =

[S]

x - cept = - K i T 1 + K Y

m

[I]

Vm

V m (app) - 1

[S] in the presence of increasing [I] and making

a Dixon plot (1/v vs [I])

1. For competitive inhibition, Ki is calculated

from

Km

x - cept = - K i T [S]

+ 1Y

17. When is an enzyme kinetically perfect?

1. kcat/Km for a kinetically perfect enzyme 108

M-1.s-1

18. What does kinetic perfection really mean?

1. It means that the enzyme is so catalytically

active that when the ES complex forms,

substrate is immediately converted to product

or to an intermediate species thereby

preventing the ES complex from dissociating

back to E and S.

19. There are a great many equations in this chapter.

Do I need to know them all?

1. No. They are provided to show how our

understanding of this field develops from basic

concepts. You should, however, try to

understand the derivations of the many

solutions presented.

73

allow you to analyze the data you may collect

in the research setting.

20. Which equations should I learn?

1. The equation for reversible substrate binding to

an enzyme:

E+S

kf

kr

ES

2. Kd is kr/kf and has units of M

3. Kd = 1/Keq

2. The equations for Vm and Bmax

1. Vm = kcat[E]t

2. Bmax =[E]tn

3. The Michaelis-Menten equation

Vm [S]

v = K + [S]

m

74

S ECTION 3

9.

Michaelis-menten equation for enzyme catalysis?

Formative self-evaluation

questions

enzyme-mediated catalysis?

11. What is Km?

by answering the following questions:

1.

binding?

2.

What is Kd?

3.

analysis of equilibrium ligand binding dier from

Kd obtained from pseudo-first-order analysis of

the time course of ligand binding to an enzyme?

4.

known?

5.

Bmax and Kd?

6.

today and why?

7.

binding?

8.

Vm and Km for enzyme-catalyzed reactions?

13. Which of these methods is used most commonly

today and why?

14. How is Vm related to [E]t?

15. How are Km and Kd related?

16. When is Km almost identical to Kd?

17. When is Km > Kd?

18. How and why does a competitive inhibitor aect

Km and Vm?

19. If you were to linearize your v vs [S] data to

illustrate the eects of a competitive inhibitor on

your enzyme, how would the inhibitor aect the

slope, y-and x intercepts of a Hanes-Wolf plot?

20. How and why does a noncompetitive inhibitor

aect Km and Vm?

21. If you were to linearize your v vs [S] data to

illustrate the eects of a noncompetitive inhibitor

on your enzyme, how would the inhibitor aect

75

plot?

22. How and why does an uncompetitive inhibitor

aect Km and Vm?

23. If you were to linearize your v vs [S] data to

illustrate the eects of an uncompetitive inhibitor

on your enzyme, how would the inhibitor aect

the slope, y-and x intercepts of a Hanes-Wolf plot

vs a Lineweaver-Burk plot?

24. Describe 2 methods for calculating Ki for inhibition

of an enzyme by competitive, noncompetitive and

uncompetitive inhibitors.

25. When can you state that an enzyme has achieved

kinetic perfection?

26. What does kinetic perfection imply in terms of

rates of catalysis vs ES dissociation to E and S?

76

S ECTION 4

Key to formative

evaluations

1. The equation is:

sites per enzyme.

5. The methods are:

1. [S]b vs [S] data can be fitted directly by

nonlinear regression analysis to yield Bmax and

Kd.

2. [S]b vs [S] data can be linearized in the

following ways to obtain Bmax and Kd from the

intercepts and slope obtained from linear

regression analysis of the plots:

1. Lineweaver-Burk (1/[S]b vs 1/[S]) gives

B max [S]

[S] b = K + [S]

d

2. Kd is that [S] at which [S]b is Bmax/2. It is also

known as the dissociation constant of the ES

complex.

3. Kd obtained from Michaelis-Menten and from

pseudo-first-order analsis of binding kinetics

should be identical. The advantage of the latter

analysis is that you also obtain kf and kr. The

advantage of the former analysis is that you do not

have to perform complex time-course studies.

1. Kd = -1/x-intercept

2. Bmax = 1/y-intercept

2. Hanes-Wolf ([S]/[S]b vs [S]) gives

1. Kd = -x-intercept

2. Bmax = 1/slope

3. Scatchard ([S]b/[S] vs [S]b) gives

1. Kd = -1/slope

2. Bmax = x-intercept

6. Curve-fitting to the Michaelis-Menten equation by

direct non-linear regression analysis is the

77

errors at low [S] where experimental error (signal to

noise) is frequently greatest. Todays computers

are more than adequate to perform this analysis.

7. A competitive inhibitor increases Kd for substrate

binding but leaves Bmax unchanged.

8. Ki is Kd for inhibitor binding to the enzyme.

9. We assumed:

ways to obtain Vm and Km from the intercepts

and slope obtained from linear regression

analysis of the plots:

1. Lineweaver-Burk (1/v vs 1/[S)] gives

1. Km= -1/x-intercept

2. Vm = 1/y-intercept

2. Hanes-Wolf ([S]/v vs [S]) gives

1. Km = -x-intercept

i.e. ES breaks down directly to E + P.

2. Vm = 1/slope

S and E to form ES does not significantly aect

free [S].

10. The equation is:

Vm [S]

v = K + [S]

m

direct non-linear regression analysis is the

preferred method because inverting data amplifies

errors at low [S] where experimental error (signal to

noise) is frequently greatest. Todays computers

are more than adequate to perform this analysis.

14. Vm = [E]t kcat

regression analysis to yield Vm and Km.

78

Vm unchanged because binding of the inhibitor and

S are mutually exclusive.

19. A Hanes-Wolf plot of v vs [S] data in the absence

or presence of a competitive inhibitor will show

that inhibitor makes the x-intercept (-Km) more

negative but leaves the slope (1/Vm) unchanged.

20. A noncompetitive inhibitor reduces Vm but leaves

Kmapp unchanged because inhibitor binding blocks

catalysis but does not aect substrate binding.

21. A Lineweaver-Burk plot of v vs [S] data in the

absence or presence of a noncompetitive inhibitor

will show that inhibitor increases the y-intercept (1/

Vm), leaves the x-intercept (1/-Km) unchanged and

increases the slope (Km/Vm).

22. An uncompetitive inhibitor reduces Vm and Kmapp

because I can bind only to the ES complex but the

ESI complex is inactive. Thus S reduces Kd for I

binding and I reduces Vm.

23. A Hanes-Wolf plot of v vs [S] data in the absence

or presence of an uncompetitive inhibitor will show

that inhibitor makes the x-intercept (-Km) more

positive, increases the slope (1/Vm) and leaves the

y-intercept (Km/Vm) unchanged.

absence or presence of an uncompetitive inhibitor

will show that inhibitor increases the y-intercept (1/

Vm), makes the x-intercept (1/-Km) more negative

and leaves the slope (Km/Vm) unchanged.

24. If we obtain Vm and Km for a reaction in the

presence or absence on an inhibitor, Ki can be

calculated in the following way:

1. For competitive inhibition, Ki is calculated as

[I]

Ki = K

m (app)

Km - 1

2. For noncompetitive and uncompetitive

inhibition, Ki is calculated as

Ki =

[I]

Vm

V m (app) - 1

and vary the inhibitor concentration [I], we can then

make a Dixon plot (1/v vs [I]) and obtain Ki as:

1. For competitive inhibition, Ki is calculated from

[S]

x - cept = - K i T 1 + K Y

m

79

as

x - cept = - K i

3. For uncompetitive inhibition, Ki is calculated

from

Km

x - cept = - K i T [S]

+ 1Y

26. An enzyme is said to have achieved kinetic

perfection when the ratio kcat/Km 108 M-1.s-1

27. This means that kp >> kr, Km >> Kd and the ES

complex never has an opprtunity to dissociate to E

+ S. The enzyme is said to be diusion-limited.

80

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