The Kinetics of Enzyme - Catalyzed Reactions

Introduction

• A breakthrough in enzymology occurred in 1897 when Buchner first

extracted active enzyme from living cells.
• Buchner work provided two major contribution
• It showed that catalysts at work in a living organism could also
function completely independently of any life process.
• Isolation and purification of enzyme
 Nomenclature of enzymes

• According to the International union Of Biochemistry an enzyme

name has two parts:
-First part is the name of the substrates for the enzyme.
-Second part is the type of reaction catalyzed by the enzyme.
This part ends with the suffix “ase”.
Example: Lactate dehydrogenase
Nomenclature of enzymes

EC number
• Enzymes are classified into six different groups according to the reaction being catalyzed.
• The nomenclature was determined by the Enzyme Commission in 1961 (with the latest
update having occurred in 1992), hence all enzymes are assigned an “EC” number.
• The classification does not take into account amino acid sequence (ie, homology), protein
structure, or chemical mechanism.
• EC numbers are four digits, for example a.b.c.d, where “a” is the class, “b” is the subclass,
“c” is the sub-subclass, and “d” is the sub-sub-subclass.
• The “b” and “c” digits describe the reaction, while the “d” digit is used to distinguish
between different enzymes of the same function based on the actual substrate in the reaction.
• Example: for Alcohol: NAD+oxidoreductase EC number is 1.1.1.1
 Naming Enzyme

• The name of an enzyme identifies the reacting substance

- usually ends in –ase
• For example, sucrase catalyzes the hydrolysis of sucrose
• The name also describes the function of the enzyme
• For example, oxidases catalyze oxidation reactions
• Sometimes common names are used, particularly for the digestion enzymes such as
pepsin and trypsin
• Some names describe both the substrate and the function
• For example, alcohol dehydrogenase oxides ethanol
 Classification of enzymes

Enzymes are often classified by placing them in categories according to

the reactions that they catalyze:
1. Oxidoreductase : Oxidation-reduction
2. Transferase : Transfer groups of atoms
3. Hydrolase : Hydrolysis
4. Lyase : Add atoms/remove atoms to/from a double bond
5. Isomerase : Rearrange atoms
6. Ligase : Use ATP to combine molecules
 Enzyme as a Biological catalyst
• Enzymes are proteins that increase the rate of reaction by lowering the
energy of activation
• They catalyze nearly all the chemical reactions taking place in the cells of
the body
• Enzymes have unique three-dimensional shapes that fit the shapes of
reactants (substrates)

- The uncatalyzed reaction has a large activation

energy, Eact,
- In the catalyzed reaction, the activation energy has
been lowered significantly increasing the rate of the
reaction
How can an enzyme reduce the activation energy?
(1) Binding to the substrate can be done such that the formation
of the transition state is favored
(2) Orientation and positioning of substrate(s)
(3) Bonds in the substrate can be ‘activated’ by functional groups
in the catalytic site
 Characteristics of enzymes

• Another distinguishing characterstics of enzymes is their frequent need for

cofactor.
• A cofactor is a nonprotein compound which combine with an otherwise
inactive protein ( the apoenzyme) to give catalytically active complex.
• This complex is called holoenzyme by biochemists, in simply term it is an
enzyme.
• Two distinct varieties of cofactor exist.
• Metal cofactor
• Complex organic molecule called a coenzyme may serve as a cofactor.
Example :FAD, NAD and Coenzyme A (CoA)
Enzyme containing or requiring metal ion as cofactor

Ions Enzyme
alpha-amaylase, collagenase, lipase, micrococcal
Ca2+ nuclease
Co2+ glucose isomerase (also require magnesium)
Cu2+ (Cu+) galactose oxidase, tyrosinase, laccase
Fe2+ (Fe3+) catalase, cytochromes, peroxidase
Mg2+ Deoxyribonuclease ( bovine pancrease), hexokinase,
Mn2+ Arginase, ribonucleotide reductase
Na+ plasma membrane ATPase ( also require K+ and Mg2+)
alcohol dehydrogenase, alkaline phosphatase,
Zn 2+ carboxypeptidase
 Enzyme specificity

• Enzymes have varying degrees of specificity for substrates

• Enzymes may recognize and catalyze:
- a single substrate
- a group of similar substrates
- a particular type of bond
 Enzyme turnover number
• Turnover number (also termed Kcat) is defined as the maximum number of
chemical conversions of substrate molecules per second that a single catalytic
site will execute for a given enzyme concentration.

Enzyme Substrate Kcat(S-1)

Catalase H2O2 40,000,000
Carbonic anhydrase HCO3- 400,000
Acetylcholinesterase Acetylcholine 14,000
β-Lactamase Benzylpenicillin 2,000
Fumarase Fumarate 800
RecA protein (an ATPase) ATP 0.4
P450 O2 0.3
 Enzyme substrate complex and enzyme action

• These reversible reaction steps represent the steps in an enzyme catalyzed

reaction
• The first step involves formation of an enzyme-substrate complex, E-S
• E-S* is the transition state
• E-P is the enzyme-product complex

• E + S  ES*  EP  E + P
 Lock-and-Key Model
• In the lock-and-key model of enzyme action:
- the active site has a rigid shape
- only substrates with the matching shape can fit
- the substrate is a key that fits the lock of the active site
• This is an older model, however, and does not work for all enzymes
Contd…

• The protein molecule is folded in such a way that a group of reactive amino acid side chains
in the enzyme present a very specific site to the substrate.
• The reactive groups encounter in enzyme include R group of Asp, Cys, Glu, His, Lys , Met,
Ser, Thr and the end amino and carboxyl functions.
• The number of such groups near the substrate is typically 20 ( far less than the total number
of amino acid residue present) , only a small fraction of the enzyme is believed to
participate directly in the enzyme active site.
• Large enzyme may have more than one active sites.
• Many of the remaining amino acids determine the folding along a chain of amino acids (
secondary structure) and the placement of one part of the folded chain next to another
(tertiary structure), which help to create active sites itself.
• Enzymes are believed to bind substrates in especially favorable position, thereby
contributing an orientation effect, which accelerate the rate of reaction.
• Binding of substrate can change the enzyme conformation. This induced fit of enzyme and
substrate may add to the catalytic process.
 Induced-fit model

• In the induced-fit model of enzyme action:

- the active site is flexible, not rigid
- the shapes of the enzyme, active site, and substrate adjust to maximize the
fit, which improves catalysis
- there is a greater range of substrate specificity
• This model is more consistent with a wider range of enzymes
 Enzyme kinetics
• An enzyme-catalyzed reaction of substrate S to product P, can be written
E
S P
Actually, the enzyme and substrate must combine and E recycled after the
reaction is finished, just like any catalyst.
Because the enzyme actually binds the substrate the reaction can be written
as:
k1 k2
E + S ↔ ES → P + E
k -1
The simplest reaction is a single substrate going to a single product.
 Rate or velocity of the reaction depends on the formation of the ES
 The P -> ES is ignored
 The equilibrium constant Keq is based on the idea that the reaction is limited
to the formation of the ES complex and that only K1 and K-1 are involved
because the thermodynamics of the reversal of K2 cause it to be minimal
k1
Keq =
K -1
How fast an enzyme catalyzes a reaction is it's rate. The rate of the reaction is in
the number of moles of product produced per second

rate (v) = - d[S] =

d[P]
= k2 [ES]
dt dt
The dimensions of v are consequently mole per unit volume per unit time.
The reaction rate is an intensive quantity , defined at each point in the reaction mixture.
Contd..

The relationship between the concentration of a substrate and the rate of an

enzymatic reaction is described by looking at the concentration of S and v
 When the reaction is first order - the rate is dependent on [S]
 When the reaction is zero order, there is no relationship between v and S
 A second order is between 1st and 0 order, where the relationship between V
and [S] is not proportional to [S]

Initial
Velocity
(Vi or V)

[ Substrate]
Contd..

• To study enzymes, first order kinetics must be followed

• Think of the graph of [S] vs. v in this way:
 The velocity increases as the substrate concentration is increased up to
a point where the enzyme is "saturated" with substrate.
 At this point the rate of the reaction (v) reaches a maximal value and
is unaffected by further increases in substrate because all of the
enzyme active site is bound to substrate
 Michaelis-Menten Kinetics

• A mathematical model of the kinetics of single-substrate-enzyme catalyzed

reactions was first developed by V.C.R. Henri in 1902 and L. Michaelis and
M.L.Menten in 1923
• Kinetics of simple enzyme-catalysed reaction are often referred to as Michalis
Menten kinetics or saturation kinetics
 Conditions for Michaelis -Menten

Two assumptions must be met for the Michaelis-Menten equation

 Equilibrium - the association and dissociation of the substrate and enzyme is
assumed to be a rapid equilibrium and Ks is the enzyme: substrate dissociation
constant.
 Steady state - the enzyme substrate complex ES is at a constant value. That is
the ES is formed as fast as the enzyme releases the product. For this to happen
the concentration of substrate has to be much higher than the enzyme
concentration. That is why we only study the initial velocity. Later in the
reaction the substrate concentration is relatively lower and the rate of product
starts to be limited by diffusion and not the mechanism of the enzyme
 Mathematical model of the representation of the M&M eq. -
For the reaction:
k1 k2
E + S ↔ ES → P + E
k -1

1) The Michaelis constant Km is:

K-1 + K 2
Km =
K1
2) When investigating the initial rate (Vo) the Michaelis-Menten equation is:

Vma x [S]
Vo =
[S] + K m

Graphical representation is a hyperbola. Think of the difference between O2 binding of

myoglobin and hemoglobin.

 When [S] << Km, the velocity is dependent on [S]

 When [S] >> Km, the initial velocity is independent of [S]
 When [S] = Km, then Vo = 1/2 V max

See details on the derivation of the MM equation on lecture notes

●Km is a measure of the affinity of the enzyme for it's substrate and also
informs about the rate of a reaction. The binding constant is appoximated by
Km

● The reaction must be first order and [S] >> E (two assumptions)
Km tells us
 Km - relates to affinity ; Vmax relates to efficiency
 Km tell how much substrate to use in an assay
 If more than one enzyme share the same substrate, KM also will determine how to
decide which pathway the substrate will take
Vmax tells about pathways
 Rate limiting enzyme in pathway
 Km and Vmax can be used to determine effectiveness of inhibitors and activators for
enzyme studies and clinical applications
See lecture notes on
- Lineaweaver Burk plot
- Eadie-Hofstee plot
- Hanes-Woolf plot
- Kinetics of reversible reaction, two substrate reactions and cofactor
activation
 Enzyme Inhibition and Inhibition Kinetics
• Enzyme inhibitors are molecules that interfere with catalysis, slowing or halting
enzymatic reactions.
• Enzyme inhibitors are among the most important pharmaceutical agents known. For
example, aspirin (acetylsalicylate) inhibits the enzyme that catalyzes the first step
in the synthesis of prostaglandins, compounds involved in many processes, including
some that cause pain.
• The study of enzyme inhibitors also has provided valuable information about enzyme
mechanisms and has helped define metabolic pathways.
• There are two broad classes of enzyme inhibitors: reversible and irreversible
inhibitors.
• Irreversible Inhibitor - Inactivator
• Reversible Inhibitor
• Competitive inhibition
• Uncompetitive inhibition
• Non-competative
• Mixed inhibitor
 Irreversible inhibitors - inactivators.
• Irreversible inhibitors bind covalently to or destroy a functional group on an
enzyme that is essential for the enzyme’s activity.
• They also can inhibit an enzyme by forming a particularly stable noncovalent
association with the enzyme. A special class of irreversible inhibitors are the
mechanism-based (suicide) inactivators.
• These compounds are relatively unreactive until they bind to the active site of a
specific enzyme.
• A suicide inactivator undergoes the first few chemical steps of the normal
enzymatic reaction, but instead of being transformed into the normal product, the
inactivator is converted into a very reactive compound that combines irreversibly
with the enzyme.
• These inhibitors earn their name because they hijack the normal enzyme reaction
mechanism to inactivate the enzyme.
 Competitive inhibitor
• A competitive inhibitor (I) competes with
the substrate for binding to the active site of
an enzyme. While the inhibitor occupies the
active site, it prevents the binding of the
substrate to the enzyme and blocks the
reaction.
• Many competitive inhibitors are structurally
similar to the substrate and combine with the
enzyme to form an EI complex, but without
leading to catalysis.
• Competitive inhibition can be analyzed
quantitatively by steady-state kinetics. In the
presence of a competitive inhibitor, the MM
equation becomes
v
d [ P]
 k 2 [ ES ] KI 
E I
dt EI
Vmax S
' k -1 [ E ][ S ]
Km  
k1 [ ES ]
vo 
KI 
[ E ][ I ] K M  S
[ EI ]
[E0 ]  [E]  [ES ]  [EI ]  I 
  1  
The experimentally determined variable Km, the
Km observed in the presence of the competitive  KI 
inhibitor, is often called the “apparent” Km
 E I
Competitive inhibitor

KI 
EI
Vmax S
The experimentally determined variable
Km, the Km observed in the presence of
vo 
K M  S
the competitive inhibitor, is often called
the “apparent” Km

 I 
  1  
 KI 
• Because a competitive inhibitor binds reversibly
to an enzyme, the competition can be biased to
favor the substrate simply by adding more
substrate to the reaction. When [S] far exceeds [I],
the probability that an inhibitor will bind to the
enzyme is minimized and the reaction exhibits a
normal Vmax.
• However, the [S] at which V0 = 1/2 Vmax, the
apparent Km, increases in the presence of inhibitor
by the factor . This affect on apparent Km,
combined with the absence of an effect on Vmax, is
diagnostic of competitive inhibition and is readily
revealed in a double-reciprocal kinetic plot.
• The equilibrium constant for inhibitor binding,
KI, can also be obtained from these plots. Many
drugs act by competitively inhibiting enzymes
(e.g., ibuprofen and the cyclooxygenase enzymes,
COX 1 & 2).
 Uncompetitive Inhibitor

Km’
k2 d [ P]
E+S ES  P  E v  k 2 [ ES ]
+ dt
I k -1 [ E ][ S ]
'
Km  
KI
k1 [ ES ]
ESI
[ ES ][ I ]
KI 
[ ESI ]

[E0 ]  [E]  [ES ]  [ESI ]

 Uncompetitive inhibitor

(Vm /(1  [ I ] / K I ))[ S ]

v
'
( K m /(1  [ I ] / K I ))  [ S ]

Vm, app [ S ]
v
'
K m , app  [ S ]
Vm, app  Vm /(1  [ I ] / K I ) ' '
K m, app / Vm, app  K m / Vm
 Non- Competitive inhibitor

• Binds to an enzyme site

different from the active site
• Inhibitor and substrate can bind
enzyme at the same time
• Cannot be overcome by
increasing the substrate
concentration
• inhibitor binds distal to active
site
• effects enzyme rate not affinity
• binds E in E S or E
• Reversible
Non- Competitive inhibitor

d [ P] Vm [ S ]
v  k 2 [ ES ] v
dt '
(1  [ I ] / K I )( K m  [ S ])
' k-1 [ E ][ S ] [ EI ][ S ]
Km   
k1 [ ES ] [ ESI ]
[ E ][ I ] [ ES ][ I ] Vm, app [ S ]
KI 
[ EI ]

[ ESI ]
v
'
K m  [S ]
[E0 ]  [E]  [ES ]  [EI ]  [ESI ]

Vm, app  Vm /(1  [ I ] / K I ) When [I] =0, Vm, app  Vm

 Mixed Inhibitor
Mixed inhibition is when the inhibitor binds to the enzyme at a location distinct
from the substrate binding site. The binding of the inhibitor will either alter the
KM or Vmax or both.

KI 
E I
KI 
ESI
EI ESI 

Vmax S  I 

vo     1  
K M   S  K I 