Chapter 8: Basic Concepts and Kinetics

1. Chapter 8 Objectives
a. Describe how enzymes work, general properties of enzymes, and
factors that contribute to enzyme catalysis
b. Determine the class of an enzyme, given the reaction
c. Given a reaction and rate data, determine the order for each substrate
and the overall order of a reaction
d. Determine Km from Michaelis-Menten plots
e. Determine the type of enzyme inhibition from Lineweaver-Burk plots
f. Compare enzyme efficiencies given Kcat and Km for various substrates
g. Describe methods of enzyme regulation
2. Enzyme/Catalyst
a. Catalyst substance that accelerates the rate of a chemical reaction
without itself becoming permanently altered in the process
b. Every reaction within the cell is regulated by enzymes
c. Enormously effective catalysts: typically enhance rates by 10 6 to 1012
d. Very specific
e. Obey laws of thermodynamics (no effect on Keq)
f. Controlled via regulatory mechanisms
g. Transition state of reacting substrates bound in enzyme active site
h. A catalyst reduces the activation energy of a reaction
i. Enzymes alter only the reaction RATE and not the reaction equilibrium
3. Free Energy
a. For the reaction: A + B <-> C + D
b. G = Go + RT ln ( [C][D]) / ( [A][B] )
c. Where Go is the standard free energy, R is the gas constant, T is 298
K, and the brackets denote concentration in moles.
d. Keq = ([C][D]) / ([A][B])
e. Keq = e^(- Go/RT)
f. The greater Keq, the more negative Go
4. Free energy cont.
a. A reaction will occur without the input of energy, or spontaneously,
only if G is negative. Such reactions are called exergonic reactions.
b. A reaction will not occur if the G is positive. These reactions are called
endergonic reactions.
c. If a reaction is at equilibrium, there is no net change in the amount of
reactant or product. At equilibrium, G = 0.
d. The G of a reaction depends only on the free energy difference
between reactants and products and is independent of how the
reaction occurs.
e. The G of a reaction provides no information about the rate of the
reaction.
5. Transition state
a. Enzymes decrease the activation energy by facilitating formation of the
Transition state
i. S XP

b. Binding energy is the free energy released upon interaction of the

enzyme and substrate
c. Binding energy is greatest when the enzyme interacts with the
transition state, thus facilitating the formation of the transition state
d. Evidence for E-S Complex
i. Maximum velocity reached with constant concentration of
enzyme
ii. Saturation
iii. Indirect evidence

iv.
6. Active Sites of Enzymes
a. The active site is a three-dimensional cleft or crevice created by amino
cids from different parts of the primary structure
b. The active site constitutes a small portion of the enzyme
c. Active sites create unique microenvironments
d. The interactions of the enzyme and substrate at the active site
involves multiple weak interactions
e. Enzyme specificity depends on the molecular architecture at the active
site
7. Catalytic Mechanisms
a. Binding enzyme uses binding energy to increase rate of reaction
b. The enzyme brings substrate together in an orientation that facilitates
catalysis
i. Proximity and orientation (entropy reduction)
ii. Electrostatic catalysis
iii. Preferential transition state binding
iv. Induced fit
c. Specific catalytic mechanisms involving proper positioning of
catalytic groups on the enzyme that aid in bond cleavage and
formation
i. Acid-base catalysis a molecule other than water donates or
accepts a proton
ii. Covalent catalysis the active site contains a nucleophile that is
briefly covalently modified
iii. Metal ion catalysis metal ions function in a number of ways
including serving as a electrophilic catalyst

8. Induced Fit
a. Binding substrate to enzyme may stabilize a different conformation of
either the enzyme or the substrate, orientating catalytic groups on the
enzyme or promoting tighter transition state binding, and/or excluding
water
9. Six Major Classes of Enzymes
a. Oxidoreductase catalyze oxidation-reduction reactions.
i. A + B A + B
ii. Pi + glyceraldehyde-3-phosphate + NAD+ NADH + H+ + 1,3bisphosphoglycerate
b. Transferases move functional groups between molecules.
i. AX + B A + BX
c. Hydrolyases cleave bonds with the addition of water.
i. AB + H2O AOH + BH
d. Lyases remove groups from (or add to) a double bond or catalyze bond
scission involving electronic rearrangement
i. >CH-CH(-NH-R)- >C=CH- + NH2-R
ii. ATP cAMP + PPi
e. Isomerases move functional groups within a molecule.
i. A B where B is an isomer of A.
f. Ligases join (ligate) two substrate molecules
i. Ab + C AC + b
ii. Ab + cD AD + b + c
10.Enzyme Co-factors
a. Cofactors
i. Coenzymes
1. Co-substrates (weakly bound)
2. Prosthetic groups (tightly bound)
ii. Essential metal ions
1. Activator ions (weakly bound)
2. Active site ions (tightly bound)
11.Kinetics is the study of reaction rates
a. When the velocity of a reaction is directly proportional to reaction
concentration, the reaction is called a first-order reaction and the
proportionality constant has units s^-1.
b. Many important biochemical reactions are bimolecular or second-order
reactions
i. 2A -> P or A + B > P
ii. V = k[A]^2 and V = k[A][B]
iii. 1/(Ms)
12.Michaelis-Menten Model Describes the Kinetics of Many Enzymes
a. A common means of investigating enzyme kinetics is to measure
velocity as a function of substrate concentration with a fixed amount of
enzyme. Under these conditions, the velocity is called the initial
velocity or V0
b.

c. Assumptions needed to derive the Michaelis-Menten Equation

i. Rate of the reverse reaction, E + P -> ES is ignored
1. Valid because measuring initial velocities, when [P] is very
low
2. K1 (the rate constant for E + S -> ES) >>>> k-1
3. Steady-state assumption: the enzyme substrate complex
(ES) is in steady state, so [ES] remains constant as
function of time
a. Rate to form ES = rate to degrade ES
d. Equations needed to know

i.

ii.

iii.
iv.
e. Michaelis Constant

f.

i.
ii. Michaelis Constant
1. KM = [S] when V0 = Vmax
Lineweaker-Burk equations

i.

ii.

iii.
iv. This double-reciprocal equation is called the Lineweaver-Burk
equation
g. Catalytic Efficiency
i.
ii. If the enzyme concentration, [E]T, is known, then Vmax = k2[E]T
and k2 = Vmax/[E]T
iii. K2, also called kcat, is the turnover number of the enzyme, which
is the number of substrate molecule converted into product per
second
iv. Kcat/KM is a measure of catalytic efficiency
1. If [S]<<KM, we can assume that free enzyme [E] [E] T
a. The Michaelis-Menten equation can be multipulated
to yield:

i.
ii. Under these conditions, kcat/KM is a measure
of catalyric efficiency because it takes into
account boht the rate of catalysis (kcat) and
nature of the enzyme substrate interaction
(KM)
h. Biochemical reactions
i. Ordered sequential

1.
2. Ternary Complex <-> Ternary Complex
ii. Random Sequential

1.
2. Ternary Complex <-> Ternary Complex
iii. Double-displacement or Ping-Pong

1.
2. Sustitued Enzyme Intermediate <-> Substituted Enzyme
Intermediate
iv. Michaelis-Menten enzyme vs Allosteric enzyme

1.

2.
13.Enzymes can be inhibited by Specific Molecules

a. 2 Types of Inhibitors
i. Irreversible bind got enzymes very tightly, often forming a
covalent bond, and inactivate them
ii. Reversible interact more loosely with enzymes and can be
displaced
1. Competitive
2. Uncompetitive
3. Non-competitive
b. Effects of Inhibition

i.
ii. Competitive, Uncompetitive, and Noncompetitive

iii.

iv.

v.
c. Production Inhibition
i. Product is structurally similar to substrate and can bind to active
site

1.

2.
d. Irreversible Inhibitors Used to Map Active Site
i. 3 Types of Irreversible Inhibitors

1. Group-specific inhibitors
a. React with specific side chain
b. Ex: DIPF
2. Affinity label or reactive substrate analogs
a. Structurally similar to substrate
b. Covalent binds to active sites
3. Suicide inhibitor or mechanism-based inhibitors
a. Modified substrates
b. Bind as substrate
c. Catalyzed to reactive intermediate that inactivates
the enzyme thru covalent modification
14.Irreversible Inhibitors Can Be Used to Map the Active Site
a. Acetylcholinesterase + DIPF -> Inactivate + F- + H+
b. Affinity label reactivity analog
c. Bromoacetol phosphate an affinity label for triose phosphate
isomerase (TPI)
i. Triose phosphate isomerase + bromoacetol phosphate ->
Inactivate enzyme
15.Suicide or mechanism-based inhibitor
a. MonoAmine Oxdiase Inhibitor (MAOIs)
b. The suicide inhibitor removes E so that the [ES] is lower, V max is lower,
and inhibition cannot be overcome at high S 0