Enzyme and Acid

- Base Catalysis
Lecture Notes in Chem. 260
Chemical Kinetics
(Physical Chemistry II)
Joel R. Salazar, Ph.D.
Catalysis – Enzyme
Kinetics
Virtually All Reactions in Cells Are
Mediated by Enzymes

 Living systems use enzymes to accelerate and

control the rates of vitally important
biochemical reactions.
 Enzymes are the agents of metabolic function.
 Most enzymes are proteins.
 Some enzymes require cofactors or
coenzymes.
Virtually All Reactions in Cells Are
Mediated by Enzymes

Figure 13.1
Reaction profile
showing the
large free
energy of
activation for
glucose
oxidation.
Enzymes lower
ΔG‡, thereby
accelerating
rate.
13.1 What Characteristic Features Define
Enzymes ?

 Catalytic power is defined as the ratio of the enzyme-

catalyzed rate of a reaction to the uncatalyzed rate.
 Specificity is the term used to define the selectivity of
enzymes for their substrates.
 Regulation of enzyme activity ensures that the rate of
metabolic reactions is appropriate to cellular
requirements.
 Coenzymes and cofactors are nonprotein
components essential to enzyme activity.
13.1 What Characteristic Features Define
Enzymes ?
 Enzymes can accelerate reactions as much as 10 over
16

uncatalyzed rates.
 Urease is a good example:
 Catalyzed rate Uncatalyzed rate Ratio 3x104/sec
3x10-10/sec 1x1014
 Enzymes selectively recognize proper substrates over
other molecules.
 Enzymes produce products in very high yields - often
much greater than 95%.
 Specificity is controlled by structure - the unique fit of
substrate with enzyme controls the selectivity for
substrate and the product yield.
The Lock and Key Model
The Six Classes of Enzymes
Class and reaction catalyzed
1. Oxidoreductases (dehydrogenases) oxidation-reduction
reactions
2. Transferases group transfer reactions
3. Hydrolases hydrolysis reactions
4. Lyases lysis, forming a double bond
5. Isomerases isomerization reactions
6. Ligases (synthetases) joining of two substrates, uses
ATP
A Mathematical statement of the Rate
of an Enzyme-Catalyzed Reaction

 Enzyme kinetics seeks to determine the

maximum reaction velocity that
enzymes can attain and binding
affinities for substrates and inhibitors.
 Analysis of enzyme rates yields insights
into enzyme mechanisms and metabolic
pathways.
13.3 What Equations Define the Kinetics of
Enzyme-Catalyzed Reactions ?
 Simple first-order reactions display a plot of the
reaction rate as a function of reactant concentration
that is a straight line
 Enzyme-catalyzed reactions are more complicated.
 At low concentrations of the substrate, the rate is
proportional to S, as in a first-order reaction.
 At higher concentrations of substrate, the enzyme
reaction approaches zero-order kinetics.
 This behavior is a saturation effect.
13.3 What Equations Define the Kinetics of
Enzyme-Catalyzed Reactions ?
Figure 13.6
A plot of v
versus [A] for the
unimolecular
chemical
reaction, A→P,
yields a straight
line having a
slope equal to k.
As [S] increases, kinetic behavior changes
from 1st order to zero-order kinetics

Figure 13.7 Substrate saturation curve for

an enzyme-catalyzed reaction.
The Michaelis-Menten Equation is the
Fundamental Equation of Enzyme Kinetics
 Louis Michaelis and Maud Menten's theory.
 It assumes the formation of an enzyme-substrate
complex.
 It assumes that the ES complex is in rapid equilibrium
with free enzyme.
 Breakdown of ES to form products is assumed to be
slower than 1) formation of ES and 2) breakdown of ES to
re-form E and S.
 Briggs and Haldane later introduced the steady state
assumption.
The Michaelis-Menten Equation is the
Fundamental Equation of Enzyme Kinetics
k1 k2
E+S ES E+P
k-1

E = enzyme concentration.
S = Substrate concentration.
ES = Enzyme-substrate complex concentration
(noncovalent). P =
product concentration. k1 =
rate constant for formation of ES from E + S.
k-1 = rate constant for decomposition of ES to E +
S. k2 = rate constant for decomposition of ES to E
Development of the Michaelis-Menten
Equation
k1 k2
E+S ES E+P
k-1

1. The overall rate of product formation: v = k2 [ES]

2. Rate of formation of [ES]: vf = k1[E][S]
3. Rate of decomposition of [ES]:
vd = k-1[ES] + k2 [ES]
4. The steady state assumption requires that:
Rate of ES formation = Rate of ES decomposition
 Michaelis-Menten Derivation

6. In solving for [ES], use the enzyme balance to

eliminate [E]. ET = [E] + [ES]
7. k1 (ET - [ES])[S] = k-1[ES] + k2 [ES]
k1 ET[S] - k1[ES][S] = k-1[ES] + k2 [ES]
8. Rearrange and combine [ES] terms:
k1 ET[S] = (k-1 + k2 + k1 [S])[ES]
k1 ET[S]
9. Solve for [ES] = -----------------------
(k-1 + k2 + k1 [S])
 Michaelis-Menten Derivation
ET[S]
10. Divide through by k1: [ES] = -----------------------
(k-1 + k2)/k1 + [S]
11. Defined Michaelis constant: KM = (k-1 + k2) / k1
12. Substitute KM into the equation in step 10.
13. Then substitute [ES] into v = k2 [ES] from step1 and
replace Vmax with k2 ET to give:
Vmax[S]
vo = -----------
KM + [S]
The dual nature of the Michaelis-Menten
equation
Combination of 0-order and 1st-order kinetics

 When S is low, the equation for rate is 1st

order in S.
 When S is high, the equation for rate is 0-
order in S.
 The Michaelis-Menten equation describes a
rectangular hyperbolic dependence of v (rate)
on S.
Understanding Vmax

The theoretical maximal velocity

 Vmax is a constant.
 Vmax is the theoretical maximal rate of the reaction - but it
is NEVER achieved in reality.
 To reach Vmax would require that ALL enzyme molecules
are tightly bound with substrate.
 Vmax is asymptotically approached as substrate is
increased.
Understanding Km

The "kinetic activator constant"

 Km is a constant derived from rate constants.
 Km is , an estimate of the dissociation constant of E from
S.
i.) Associated with the affinity of enzyme for the
substrate( a measure of ES binding)
 Small Km means tight binding; large Km means weak binding.
ii.) Km is a measure of the substrate conc. required for effective
catalysis to occur.
 An enzyme with a high Km requires a higher substrate
conc. to achieve a given reaction velocity
The Turnover Number Defines the Activity
of One Enzyme Molecule

A measure of catalytic activity

 kcat, the turnover number, is the number of substrate

molecules converted to product per enzyme
molecule per unit of time, when E is saturated with
substrate. A measure of rate of enzyme activity.
 If the M-M model fits, k2 = kcat = Vmax/Et.
 Values of kcat range from less than 1/sec to many
millions per sec.
The Ratio kcat/Km Defines the Catalytic
Efficiency of an Enzyme
The catalytic efficiency: kcat/Km
An estimate of "how perfect" the enzyme is
 kcat/Km is an apparent second-order rate constant.
 It measures how the enzyme performs when S is low.
 The upper limit for kcat/Km is the diffusion limit - the
rate at which E and S diffuse together.
 The maximum rate of diffusion for small molecules is
109 M-1-sec-1.
Catalytic Efficiency
Sample Problem
 1.) An enzyme was observed to have the following KM

and Kcat values for three substrates ,B and C. If all

three substrates are present in the reaction mixture,
which substrate would be acted upon most rapidly?
 Substrate KM (mmol) Kcat (s-1)

 A 4 24
 B 15 37
 C 0.64 18
Turnover Number

Example calculation
 An enzyme (1.84 gm, MW 36800), in presence
of excess substrate catalyzes at a rate of 4.2
mol substrate/min. Calculate the TON. (mol
S/min/mol E)
Turnover Number
Example Calculation
 An enzyme (1.84 gm, MW 36800), in presence of excess
substrate catalyzes at a rate of 4.2 mol substrate/min.
Calculate the TON. (mol S/min/mol E)

1.84 gm
 mol E: = --------------------- = 5 x 10-5 mol E
36800 gm/mol

Vmax 4.2mol S/min

• TON = ------ = ----------------------- = 84000 min-1
Et 5 x 10-5 mol E
Michaelis-Menten Equation

Example calculation
 The rate of an enzyme catalyzed reaction is 35
mol/min at [S] = 10-4 M. KM for this substrate is
= 2 x 10-5 M.
Calculate the rate where [S] = 2 x 10-6 mol/min.
Michaelis-Menten Equation
Example calculation
 The rate of an enzyme catalyzed reaction is 35 mol/min at [S] =
10-4 M. KM for this substrate is = 2 x 10-5 M.
Calculate the rate where [S] = 2 x 10-6 mol/min.

VM [S] VM (10-4)
• v = ------------- so 35 = ---------------------
KM + [S] (2 x 10-5) + (10-4)
And VM = 1.2(35) = 42 mol/min

(42)(2 x 10-6) (84 x 10-6)

• v = -------------------------- = ------------- = 3.8 mol/min-1
(2 x 10-5) + (2 x 10-6) (22 x 10-6)
Graphical Determination of KM and VM
The Michaelis-Menten plot only permits an
estimate of Vmax, so KM is also an estimate at VM/2.
There are several graphical methods which provide a
better determination of VM and KM.
 We will focus on the Lineweaver-Burk equation which
is obtained by taking the reciprocal of the Michaelis-
Menten equation
 A Lineweaver-Burke plot is frequently referred to as a
double reciprocal plot since one plots 1/v vs 1/[S].
 The plot gives a straight line which has a slope of
KM/VM, a y-intercept of 1/VM and an x-intercept of -
1/KM.
Linear Plots Can Be Derived from the
Michaelis-Menten Equation
Be able to develop this equation

 Lineweaver-Burk:
 Begin with v = Vmax[S]/(Km + [S]) and take the
reciprocal of both sides.
 Rearrange to obtain the Lineweaver-Burk equation:

1  Km  1  1
  
v  Vmax   [ S ]  V max a straight line.
 A plot of 1/v versus 1/[S] should yield
Linear Plots Can Be Derived from the
Michaelis-Menten Equation

The Lineweaver-
Burk equation.

Figure 13.9
The Lineweaver-
Burk double-
reciprocal plot.
Eadie – Hofstee Plot
 Sample Problem

1.)The following data were gathered for the myosin –catalyzed hydrolysis
of ATP at 25oC and pH =7.0 Determine the Michaelis –Menten
constant.
Ans. m = 76.58 s ; M-M constant = 16.8 µmol /L

[ATP] µmol L- Initial rate/ µmol L-1s-1

7.5 0.067
12.5 0.095
20.0 0.119
43.5 0.149
62.5 0.185
155.0 0.191
320.0 0.195
Sample Problem
2.) Carbonic anhydrase (Mr =30KDa) catalyzes the
reversible hydration of CO2. This reaction is
important in the transport of CO2 from the tissues to
the lungs. A.) If 10 μg of pure carbonic anhydrase
catalyzes the hydration of 0.30 g CO2 in 1 min at
37oC, under optimal conditions, what is the turnover
number for carbonic anhydrase?
B.) If the km of the enzyme is 1.2 x10-2, at what
substrate conc. Will the enzyme show ¼ of its max.
velocity?
Sample Problem
3.) An enzyme was assayed at an initial conc.
of 10-5M Substrate. The Km for the substrate
is 2.0 x 10-3. At the end of 1 minute, 2 % of
the substrate had been converted to product.
A.) What % of the substrate will be converted
to product at the end of 3 minutes. What will
be the product and substrate conc. after 3
minutes. b.) what is the max. attainable
velocity (Vmax) with the enzyme conc. used.
Enzymatic Activity is Strongly
Influenced by pH

 Enzyme-substrate catalysis is greatly dependent

on pH.
 Enzymes have a variety of ionizable side chains
that determine its secondary and tertiary structure
and also affect events in the active site.
 Substrate may also have ionizable groups.

 The effects of pH may be due to effects on Km or

Vmax or both.
Enzymatic Activity is Strongly Influenced
by pH
Enzymes are usually active only over a limited range of pH

Figure 13.11 The pH activity profiles of four different enzymes.

The Response of Enzymatic Activity to
Temperature is Complex

 Rates of enzyme-catalyzed reactions generally

increase with increasing temperature.
 However, at temperatures above 50° to 60° C,
enzymes typically show a decline in activity.
 Two effects here:
 Enzyme rate typically doubles in rate for every 10°C
as long as the enzyme is stable and active.
 At higher temperatures, the protein becomes
unstable and denaturation occurs.
The Response of Enzymatic Activity to
Temperature is Complex

Figure 13.12
The effect of
temperature on
enzyme activity.
13.4 Inhibition of Enzyme Activity
Inhibition = the rate of the process is decreased in the presence
of inhibitor.
 Enzymes may be inhibited reversibly or irreversibly.

 Reversible inhibitors may bind at the active site or at some

other site.
=Noncovalent binding of the inhibitor and can always be
reversed (removal of inhibition)
 Reversible inhibitors typically change V , K or both.
M M
 There are three common types of reversible inhibition:

 Competitive

 Non-competitive

 Uncompetitive
13.4 Inhibition of Enzyme Activity
 Enzymes may also be inhibited in an irreversible manner.
The inhibitor is covalently bound to the enzyme
(frequently encountered in the action of poisons)
 Iodoacetate is an irreversible inhibitor.
 Penicillin is an irreversible inhibitor.
 Irreversible inhibitors are also called suicide inhibitors.
 Enzymes Can Be Inhibited Irreversibly

Penicillin is an
irreversible inhibitor of
the enzyme glycoprotein
peptidease, the enzyme
which catalyzes an
essential step in bacterial
cell wall synthesis.
It covalently blocks the
active site serine.
Enzyme Inhibition

Uncompetitive
Competitive I binds with ES only
I binds at the active site VM changes
VM does not change KM changes
KM changes Slope does not
change

Noncompetitive
I does not compete for the active site
but affects the catalytic event
VM changes
KM does not change
 Michaelis-Menten Equations
Uninhibited: Competitive:
Vmax[S] Vmax[S]
v = ----------- v = ------------------------
KM + [S] KM(1 + [I]/Ki) + [S]

Noncompetitive: Uncompetitive:
(Vmax/(1 + [I]/Ki)) [S] (Vmax/(1 + [I]/Ki))[S]
v = ------------------------- v = -------------------------
KM + [S] KM(1 + [I]/Ki) + [S]
Types of Inhibition
Competitive Inhibitors Compete With
Substrate for the Same Site on the Enzyme

Figure 13.13 Lineweaver-Burk plot of competitive inhibition,

showing lines for no I, [I], and 2[I].
Succinate Dehydrogenase – a Classic
Example of Competitive Inhibition

Figure 13.14 Structures of succinate, the substrate of

succinate dehydrogenase (SDH), and malonate, the
competitive inhibitor. Fumarate is also shown.
Pure Noncompetitive Inhibition – where S
and I bind to different sites on the enzyme

Figure 13.15 Lineweaver-Burk plot of pure noncompetitive

inhibition. Note that I does not alter Km but that it decreases
Vmax.
Uncompetitive Inhibition, where I
combines only with ES, but not with E

Figure 13.17
Lineweaver-Burk plot of
uncompetitive inhibition.
Note that both
intercepts change but
the slope (Km/Vmax)
remains constant in the
presence of I.
Acid – Base Catalysis
(Homogeneous)
 Homogeneous catalysis (single phase) in solution
 Typically liquid and at low temperature

 Types: 1.) Specific Catalysis (the rate of

reaction is proportional to the conc. of H+ and
OH-
2.) General Catalysis
ex. Hydrolysis of an ester
Mutarotation of Glucose
2.Acid –Base CATALYSIS

Bronsted Acid –Base reaction

HA + H2O === H3O+ + A-
acid
A- + H2O === HA + OH-
base
The rate of a catalyzed reaction is given by
v = kcat [S] where [S]
where kcat = ko + kH+[H3O+] + kOH-[OH-] + kHA[HA] + kA-[A-]
 Bronsted Acid – Base
Catalysis
 the rate constant ka for acid catalysis or kB for base
catalysis is proportional to the ionization constant K a for
the acid or Kb for the base
 kA = CA KAα
 kB = CB KB β
where: α and β have a value from 0 to 1.
Low value of α and β indicate a low sensitivity of the
catalytic constant to the strength of the catalyzing acid or
base.
Kinetics of Acid –Base
Catalysis
S + HA === SH+ + A- (k1/k-1)
SH+ + H2O === P (k2)
d[SH+]/ dt = 0;
k1 [S][HA] – k-1[SH+][A-] – k2[SH+]

d[P]/dt = k2[SH+] = k1k2[S][HA] / k-1[A-] + k2

If k2>> k-1[A]
d[P]/dt = k1[S][HA] general acid catalysis

If k2<< k-1[A- ]
d[P]/dt = k1k2[S][HA]/ k-1[A- ] = k1k2[S][H+]/ k-1Ka
Specific acid catalysis
Specific Acid –Base Catalysis
 Rate is proportional only to the concentration of H3O+/ OH- present
kH+ and kOH- >> kHA and kA-
kcat = ko + kH+[H3O+] + kOH- [OH-]

i.) if the reaction is catalyzed only by acids

kcat = ko + kH+[H3O+]
if ko ( uncatalyzed rxn) is negligible
log kcat = log k[H+] + log [H3O+]

ii.) for a reaction catalyzed only by bases

kcat = ko + kOH- [OH-]
if ko ( uncatalyzed rxn) is negligible
log kcat = log k[OH-] Kw + pH
kcat is determined by measuring the rate in a solution of constant
ionic strength (conc.) over a wide range of pH.
 Effect of pH on the rate of some
acid –base catalyzed reaction
1.) Hydrolysis of ester (subject to both acid and
base)
2.) Inversion of sugar (catalyzed by acid only)
3.) Aldol condenzation of acetaldehyde ( catalyzed
by bases only)
Catalysts Open Up New Reaction Pathways
O−

C + H2O
CH2 CH3
OH− −OH−

Base catalyzed
O OH
rate = k[OH−][acetone]
C C
CH3 CH3 CH2 CH3

propanone propenol
‡ ‡

propenol
intermediate
propanone
Catalysts Open Up ‡New Reaction
‡ Pathways

propenol
different
propanone intermediate
propenol
O OH
propanone rate = k[H3O+][acetone]
C C
Acid catalyzed
CH3 CH3 CH2 CH3

H3O+ −H3O+
OH

C
+
CH3 CH3

+ H2O
General Acid –Base Catalysis
kcat = ko + kH+[H3O+] + kOH-[OH-] + kHA[HA] + kA-[A-]
equation representing the general acid –base catalysis

If the solution is buffered (the rate is not affected by H 3O+

and OH-)
kcat = kHA[HA] + kA-[A-]

 equation containing a weak acid [HA] and its conjugate

base.
 General Acid –Base Catalysis
How to determine kHA and kA
The reaction is studied at two different pH values
Where [HA]/[A-] is a known constant say X1and X2

kcat1 = kHA [HA] + kA- [HA] / x1 where : [HA] / [A-] = x1

[A-] = [HA] / x1
kcat2 = kHA[HA] + km
A [HA]
-=k
/ xkA2-/x1
HA + and
kHA + kA-/x2
kcat

[HA]
Sample Problem
1.) The mutarotation of glucose is first order in glucose concentration and is
catalyzed by acids (A) and bases (B). The first order rate constant may be
expressed by an equation of the type that is encountered in reactions with
parallel paths:
k = ko + kH+[H+] + kA[A] + kB[B]
where ko is the first – order rate constant in the absence of acids and bases.
The following data were obtained at 180C in a medium containing 0.02
mol/L sodium acetate and various concentrations of acetic acid.
[CH3COOH] (M) 0.020 0.105 0.199
k (10-4/min) 1.36 1.40 1.46
Calculate ko and kA. The term involving kH+ is negligible under these
conditions.

Answer: 1.35 x 10-4/m and 5.5 x 10-5L/molmin

3. Autocatalysis and Chemical Oscillators
Oscillating Reactions
Autocatalysis
 Reaction is accelerated by the product

 e.g A ==== P (k)

 The rate law involves product
(the reaction rate increases as [P]
increases.)

 Rate = k [A][P]
Autocatalysis
reactions where the product “B” is one of the reactants.

In ( [B][Ao] / [A][Bo] ) = ([Ao] + [Bo]) kt

Oscillating Reactions
 The concentrations of reactants, intermediates, and
products of certain chemical reactions can vary
periodically in either in space or time.

 If we make up a mixture of potassium bromate, malonic

acid and a cerium (IV) salt in acidic solution and watch
we see:
Oscillating Reactions
 This is an example of autocatalysis.

 The best known chemical oscillator is the Belousov-Zhabotinski

reaction: (the oxidation of malonic acid by bromate ion catalyzed by
Cerium ions
 2H+ + 2BrO3- + 3CH2(CO2H)2 == 2 Br CH(CO2H)2 + 3CO2 +
4H2O
 When this reaction is catalyzed with Ce3+, the concentration of the
intermediate Br - and the ratio [Ce4+]/[Ce3+] oscillate. The catalyzed
reaction involves about 18 steps and 21 different chemical species.