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Kinetics
Enzymes are commonly named by adding the suffix ase to the substrate the
enzyme converts or a phase that describes the reaction catalyzed by the enzyme.
Lehninger 2000 Table 8.3
Enzyme Commission Number
An example:
The four-part enzyme classification number is a series of 4 numbers
separated by periods.
Help can be in the form cofactors which are either one or more
inorganic ions or a complex organic or metalloorganic molecules
called a coenzyme.
Cofactors table
Perncious anemia
Pellagra
Megaloblastic anemia
Beriberi
What do enzymes do?
Thermodynamics Again?
General
Base
Catalyzed base
Acid catalysis
decarboxylation
covalent bond electron withdrawal
Transition state
Typical Stages:
1. Nucleophilic Rx between catalyst & substrate to form covalent bond.
2. Withdrawal of electrons from reaction center by the (now) electrophilic catalyst.
3. Elimination of the catalyst (reverse of first step)
Voet, Voet & Pratt 2013Fig 11.11
Covalent Catalysis
Covalent Catalysis (nucleophilic catalysis) definition:
accelerates the reaction via the transient formation of a
catalyst-substrate covalent bond. The typical way this
happens is a nucleophile group on the enzyme forms a
covalent bond with an electrophile group on the substrate.
Nearly 1/3 of all enzymes utilize a metal ion for their catalytic function.
Voet, Voet & Pratt 2008 Fig 11.13
Electrostatic Catalysis
They bring substrate into contact with catalytic groups & multiple
substrates with each other. ~ 5-fold boost
They bind their substrates in the proper orientation to promote the
reaction ~100-fold boost
They freeze out relative translational and rotational motion of catalytic
groups and substrates in transition state little relative motion of
catalytic groups ~107-fold boost
ES
An enzyme may bind the transition state with greater affinity than
the substrates or products This increases the molecules in the
transition state, thus proportionally increasing the reaction rate
Lehninger 2013 Figure 11.15
Kinetics
The study of reaction rates
Goals -Kinetics
Know the difference between 1st and 2nd order reactions and their half-lives.
Know the nomenclature for enzyme kinetics.
Understand the difference between rapid-equilibrium and steady-state approaches to
enzyme kinetics.
Know the Michaelis-Menten equation and how to plot it.
Understand the concepts of Km and Vmax.
Understand the Lineweaver-Burke Plot, be able to plot it and extract kinetic
constants. Be aware that there are other types of kinetic plots
Understand enzyme reaction reversibility and how that affects the kinetic equation.
Understand basic types of multisubstrate enzymes (sequential and random, BiBi and
ping-pong) and how that effects their kinetic equations.
Know the various types of enzyme inhibition, how their L-B plots look, and how to
get quantitative information from those plots.
Understand how pH and temperature (Arrhenius eq.) affect enzyme activity
Setting Up A Kinetic Analysis
1. Write differential equations for each enzyme species.
2. Write the velocity equations.
3. Substitute in the differential equations the expression for
the enzyme species.
Internet Explorer Demonstration
Chemical Kinetics Elementary Rxs
Reaction Stoichiometry
A P substance A; product P
A I1 I2 P intermediates In
Rate Constant k
The proportionality constant at constant temperature describing the
rate of an elementary reaction. k is proportional to the frequency at which
the reacting molecules come together.
Reaction Velocity
The instantaneous rate of product appearance or reactant
disappearance.
Reaction Order
The number of molecules that must simultaneously come together
(collide) to generate a product, i.e. the molecularity
Reaction Order First Order
First Order Reaction A P
First Order Equation:
d[P] d[A]
v= dt = - dt = k[A]
1 1
[A] = [Ao] + kt Half-life : t = 1/k[Ao]
A+B P
Second Order Equation:
d[A] d[B]
v = - dt = - dt = k[A][B] k is in M-1 S-1 units
Pseudo first order (for B if [A] >>[B] & vice versa)
Rate Equation
Describes reaction progress as a function of time.
Derivation: (where t = time)
d[A]
[A] = d ln[A] = - k dt Integrate from [Ao] to [A]
[A] t
[ Ao] d ln [A] = - k 0 dt
k t = ln [A0]/2
[Ao]
t = ln 2/k = 0.693 / k Half Life: t
Second Order
t = 1/(k [Ao])
Enzyme Kinetics
v = reaction velocity
vo = initial reaction velocity, when [P] ~ 0
Two Approaches to Enzyme
Kinetics
Problem: One does not know the concentration of ES during a
reaction. Therefore, one has to make certain assumptions for
calculations
v = k2 ([S]/Ks)[E]
[E]T [E] + ([S]/Ks)[E] (6)
Henri-Michaelis-Menten III
E + S k1
k-1 ES k2
E + P
Cross multiple by k2 and cancel out [E] (on right side)
v = ([S]/Ks)
k2 [E]T 1 + ([S]/Ks) (6)
v = ([S]/Ks)
Vmax 1 + ([S]/Ks) (8)
v = [S]
Vmax Ks + [S] (9)
A Simple Enzyme Reaction and the
Steady-State (Progress Curves)
Michaelis-Menten equation
o
12-1b_MichaelisMenten\MichaelisMenten.htm
Significance of Km
Operational Definition: The substrate concentration at which the
reaction velocity is half maximal, i.e. when Km = [S] then vo = Vmax
When [S] << Km very little ES is formed and then [E] [E]T
Vo (k2/Km)[E]T [S] (kcat/Km) [E] [S]
Under these conditions (kcat/Km) is a measure of the enzymes catalytic
efficiency since this apparent second order rate constant (depends on BOTH [E]
and [S]) the rate of the reaction depends on how often E and S encounter each
other in solution.
Most efficient enzymes have this ratio near the
diffusion-controlled limit: 108 109 M-1s-1 kcat/Km k1
Kinetic Data Analyses
Michaelis-Menten
Michaelis-Mentenequation
equation
o
Lineweaver-Burke Plot
y = (m) x + b
1 Km 1 + 1
vo Vmax [S] Vmax
y = m * x + b
v Vmax
Km* v +
[S] Km
E
A+ B P + Q
Sequential Mechanisms: Types
Ordered
Bi-Bi
Random
Bi-Bi
V [A][B]
Vmax Kma[A] + Kmb[B] + [A][B]
Reversible Types:
Competitive
Uncompetitive
Mixed (Noncompetitive)
Inactivator - Any substance that irreversibly binds to an enzyme.
(appears similar to noncompetitive inhibition.)
Competitive Inhibition
Kmapp = Km [1 + ([I]/Ki)]
KI = [E][I] / [EI]
v = k2 [ES] vmax = k2[E]T
v k2 [ES]
[E]T [E] + [ES] + [EI]
v ([S]/Km) [E] Voet, Voet & Pratt 20 Figure 12.8
Matthews et al.1999 Figure 11.20 Voet, Voet & Pratt 2013 Figure 12.7
Uncompetitive Inhibition: M-M enzyme in
k1
k-1 k 2
L-B Plot
KI = [ES][I] / [ESI]
v = k2 [ES] vmax = k2[E]T
v k2 [ES]
[E]T [E] + [ES] + [ESI] Voet, Voet & Pratt 2013 Fig 12.9
log A
k = Ae-Ea/RT Slope
ln k = ln A Ea/RT or -Ea/2.303R
0.4 mM
Redox