Reactionkinetics:1storderreactions

k1
A B (+ C)

Decay reactions, like radio-activity;

SN1 reactions
d[A] [A]
Rate: - = k1[A]
dt
d[A]
Rewriting: - = k1dt t
[A]

t t
Integration gives: 1 d[A] ktdt
0 [A] 0
[ A ]t
So: ln[A]t ln[A]0 = -kt or: ln = -kt
[ A]0
 The time for half of the
reactant initially present to
decompose, its half-time or
half-life, t1/2,isaconstantand
hence independent of the
initial concentration of
reactant.

By substituting the relationship [A] = [A0] / 2

when t = t1/2intoln[A]=ln[A]0kt
andrearranging:

t1/2=ln2/k=0.693/k
 2A P
Second-order reaction

The half-time for a second order reaction is expressed t 1/2 = 1/k [A]0 and therefore, in contrast to a

first order reaction depends on the initial reactant concentration.

A+B P

Here, the reaction is said to be first order in A and first order in B.

Unimolecular and bimolecular reactions are common.

Termolecular reactions are unusual because the simultaneous
collision of three molecules is a rare event. Fourth and higher
order reactions are unknown.
Enzyme Kinetics

-fructofuranosidase: Sucrose + H2O glucose + fructose

When [S] [E] : the rate is zero order with respect to sucrose
The Michaelis-Menten Equation

This equation cannot be explicitly integrated, however, without simplifying assumptions,

two possibilities are

1. Assumption of equilibrium. Leonor Michaelis and Maud Menten, building on the

work of Victor Henri, assumed that k-1 k2, so that the first step of the reaction reaches
equilibrium.

Ks is the dissociation constant of the first step in the enzymatic reaction

The Michaelis-Menten Equation

1. Assumption of steady-state. Figure illustrates the progress curves of the various

participants in reaction

2. under the physiologically common conditions that substrate is in great excess over
3. Enzyme ([S] [E]).

ES maintains a steady state and [ES] can be

treated as having a constant value:

The so called steady state assumption, a more

general condition than that of equilibrium, was
first proposed in 1925 by G. E. Briggs and B. S.
Haldane
The Michaelis-Menten Equation

Letting [E] = [E]T - [ES] and rearranging yields

The Michaelis constant, KM ,

is defined as

Solving for [ES],

The Michaelis-Menten Equation

The expression of the initial velocity (v0) of the reaction, the velocity
at t=0, thereby becomes

The maximal velocity of a reaction, Vmax occurs at high substrate concentrations when
the enzyme is saturated, that is, when it is entirely in the ES form

Therefore, combining the last two equations, we obtain:

This expression, the Michaelis-Menten equation, is the basic equation of enzyme

kinetic.
 Significance of the Michaelis Constant
The Michaelis constant, KM, has a simple operational definition. At the substrate
concentration at which [S] = KM, this equation

yields v0 = Vmax/2 so that

KM is the substrate concentration at which the reaction velocity is half maximal

 Lineweaver-Burk or double-reciprocal plot

Analysis of Kinetic Data

S >> Km
vi=Vmax
Vmax= k2Et
 Vmax= 10 M/seg Km=10 x10-5 M
Si en el ensayo se usaron 5mg/L de preparacin enzimtica, entonces:
v= Vmax = k2 ET k2= 10/5 = 2 moles/mg seg
Qu predicciones podemos hacer a partir de esta informacin?
Significance of the Michaelis Constant

The magnitude of KM varies widely with the identity of the enzyme and the nature of the substrate. It
is also a function of temperature and pH. The Michaelis constant can be expressed as

Since Ks is the dissociation constant of the Michaelis complex, as Ks decreases, the enzymes affinity
for substrate increases. KM in therefore also a measure of the affinity of the enzyme for its substrate,
provided k2/k1 is small compared to Ks, that is k2 k-1 so that the ES P reaction proceeds more
slowly than ES reverts to E + S

kcat/KM Is a Measure of Catalytic Efficiency

We can define the catalytic constant, kcat, of an enzyme as

This quantity is also known as the turnover number of an enzyme because it is the number of
reaction processes (turnovers) that each active site catalyzes per unit time.
 Turn Over Numbers of Enzymes

Enzymes Substrate kcat (s-1)

Catalase H2O2 40,000,000
Carbonic anhydrase HCO3- 400,000
Acetylcholinesterase Acetylcholine 140,000
b-Lactamase Benzylpenicillin 2,000
Fumarase Fumarate 800
RecA protein (ATPase) ATP 0.4
The number of product transformed from substrate
by one enzyme molecule in one second
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.263
kcat/KM Is a Measure of Catalytic Efficiency

When [S] KM, very little ES is formed. Consequently, [E] [E]T, so

reduces to a second-order rate equation:

The quantity kcat/KM is a measure of an enzymes catalytic efficiency.

There is an upper limit to the value of k cat/KM : It can be not greater than k 1; that is, the decomposition of ES
to E + P can occur no more frequently than E and S come together to form ES. The most efficient enzymes
have kcat/KM values near to the diffusion-controlled limit of 10 8 to 109 M-1.s-1
Chymotrypsin Has Distinct kcat / Km to Different Substrates

O R O

=

H3CCNCCOCH3

H H
R= kcat / Km
Glycine H 1.3 10-1
Norvaline CH2CH2CH3 3.6 102

Norleucine CH2CH2CH2CH3 3.0 103

Phenylalanine CH2 1.0 105

(M-1 s-1)
Adapted from Mathews et al (2000) Biochemistry (3e) p.379
 Al iniciar:
t = 0, S = So

A cualquier tiempo:
T=t S=S
X = (So-S)/So

- dS/dt = vi = So dX/dt
TemperatureDependenceofEnzymes

Asisthecasewithmostreactions,anincreasein
temperaturewillresultinanincreaseinkcatforan
enzymaticreaction.
Fromgeneralprinciples,itcanbedeterminedthat
therateofanyreactionwilltypicallydoublefor
every10Cincreaseintemperature.
Manyenzymesdisplaymaximumtemperatures
around40C,whichisrelativelyclosetobody
temperature.
Thereareenzymesthatareisolatedfrom
thermophilicorganismsthatdisplaymaximaaround
100C,andsomethatareisolatedfrom
psychrophilicorganismsthatdisplaymaximaaround
10C.
 Enzyme Inhibition (Mechanism)

I Competitive I Non-competitive I Uncompetitive

Substrate E
X
Cartoon Guide

S S E I
S

E S I I
I
Compete for S I
Inhibitor active site Different site

E + S
ES E + P E + S ES E + P E + S
ES E + P
Equation and Description

+ + + +
I I I I

EI EI + S EIS EIS
[I] binds to free [E] only, [I] binds to free [E] or [ES] [I] binds to [ES] complex
and competes with [S]; complex; Increasing [S] can only, increasing [S] favors
increasing [S] overcomes
not overcome [I] inhibition. the inhibition by [I].
Inhibition by [I].

Juang RH (2004) BCbasics

 Competitive Inhibition
Product Substrate Competitive Inhibitor

Succinate Glutarate Malonate Oxalate

C-OO- C-OO- C-OO- C-OO- C-OO-

C-H H-C-H H-C-H H-C-H C-OO-
C-H H-C-H H-C-H C-OO-
C-OO- C-OO- H-C-H
C-OO-

Succinate Dehydrogenase

Adapted from Kleinsmith & Kish (1995) Principles of Cell and Molecular Biology (2e) p.49
 Sulfa Drug Is Competitive Inhibitor
Domagk (1939)

Para-aminobenzoic acid (PABA)

Bacteria needs PABA for

H2N- -COOH the biosynthesis of folic acid

Folic Tetrahydro-
Precursor
acid folic acid

Sulfa drugs has similar

H2N- -SONH2 structure with PABA, and
inhibit bacteria growth.
Sulfanilamide
Sulfa drug (anti-inflammation)

Adapted from Bohinski (1987) Modern Concepts in Biochemistry (5e) p.197

EnzymeInhibition

Many substances alter the activity of an enzyme by reversibly combining with it in a way
what influence the binding of substrate and/or its turnover number. Substances that
reduce an enzymes activity in this way are known as inhibitors

CompetitiveInhibition
A substance that competes directly with a normal substrate for an enzymes substrate-
binding site is known as a competitive inhibitor.

Here it is assumed that I, the inhibitor, bind

reversibly to the enzyme and is in a rapid
equilibrium with it so that

And EI, the enzyme-inhibitor complex, is

catalytically inactive. A competitive inhibitor
therefore reduces the concentration of free
enzyme available for substrate binding.
EnzymeInhibition

CompetitiveInhibition

This is the Michaelis-Menten equation

that has been modified by a factor, ,
which is defined as

Is a function of the inhibitors concentration and

its affinity for the enzyme. It cannot be less than 1.
EnzymeInhibition

CompetitiveInhibition

Recasting in the double-reciprocal form yields

A plot of this equation is linear and has a slope of KM/Vmax, a 1/[S] intercept
of -1/ KM, and a 1/v0 intercept of 1/ Vmax
EnzymeInhibition

UncompetitiveInhibition
In uncompetitive inhibition, the inhibitor binds directly to the enzyme-substrate complex
but not to the free enzyme

In this case, the inhibitor binding step has the dissociation constant

The uncompetitive inhibitor, which need not resemble the substrate, presumably distorts
the active site, thereby rendering the enzyme catalytically inactive.
EnzymeInhibition
UncompetitiveInhibition

The double-reciprocal plot consists of a family of parallel lines with slope KM/Vmax, 1/v0
intercepts of /Vmax and 1/[S] intercept of -/KM
EnzymeInhibition
MixedInhibition(noncompetitiveinhibition)

A mixed inhibitor binds to enzyme sites that participate in both substrate binding and
catalysis. The two dissociation constants for inhibitor binding

Double-reciprocal plots
consist of lines that have
the slope KM/Vmax, with a
1/v0 intercept of /Vmax and
1/[S] intercept of -/ KM
 Enzyme Inhibition (Plots)

I Competitive I Non-competitive I Uncompetitive

Vmax Vmax Vmax
vo vo
Direct Plots

Vmax Vmax
I I I

Km Km [S], mM Km = Km [S], mM Km Km [S], mM

Vmax unchanged Vmax decreased
Both Vmax & Km decreased
Km increased Km unchanged
Double Reciprocal

1/vo I 1/vo I 1/vo

I
Two parallel
Intersect lines
at Y axis 1/ Vmax Intersect 1/ Vmax 1/ Vmax
at X axis

1/Km 1/[S] 1/Km 1/[S] 1/Km 1/[S]

Juang RH (2004) BCbasics

BisubstrateReactions

Almost all of these so called bisubstrate reactions are either transferase reactions in
which enzyme catalyzed the transfer of a specific functional group, X, from one of
the substrates to the other:

or oxidation-reduction reactions in which reducing equivalents are transferred

between two substrates.

Sequential Reactions

Reactions in which all substrates

must combine with the enzyme
before a reaction can occur and
products be released are known as
Sequential reactions
BisubstrateReactions
Sequential Reactions

Ordered bisubstrate reaction

A and B : substrates in order that they add to the
enzyme
P and Q : products in order that they leave the
enzyme

Random bisubstrate reaction

Ping Pong Reactions

Group-transfer reactions in which one or

more products are released before all
substrates have been added are known as
Ping Pong reactions