Chapter 8: Basic Concepts and Kinetics

1. Chapter 8 Objectivesa. Describe how enzymes work, general properties of enzymes, and factors that contribute

to enzyme catalysisb. Determine the class of an enzyme, given the reactionc. Given a reaction and rate data, determine the order for each substrate and the overall

order of a reactiond. Determine Km from Michaelis-Menten plotse. Determine the type of enzyme inhibition from Lineweaver-Burk plotsf. Compare enzyme efficiencies given Kcat and Km for various substratesg. Describe methods of enzyme regulation

2. Enzyme/Catalysta. Catalyst – substance that accelerates the rate of a chemical reaction without itself

becoming permanently altered in the processb. Every reaction within the cell is regulated by enzymesc. Enormously effective catalysts: typically enhance rates by 106 to 1012

d. Very specifice. Obey laws of thermodynamics (no effect on Keq)f. Controlled via regulatory mechanismsg. Transition state of reacting substrates bound in enzyme active siteh. A catalyst reduces the activation energy of a reactioni. Enzymes alter only the reaction RATE and not the reaction equilibrium

3. Free Energya. For the reaction: A + B <-> C + Db. ΔG = ΔGo + RT ln ( [C][D]) / ( [A][B] )c. Where ΔGo is the standard free energy, R is the gas constant, T is 298 K, and the brackets

denote concentration in moles. d. K’eq = ([C][D]) / ([A][B])e. K’eq = e^(- ΔGo/RT)f. The greater K’eq, the more negative ΔGo’

4. Free energy cont.a. A reaction will occur without the input of energy, or spontaneously, only if ΔG is

negative. Such reactions are called exergonic reactions.b. A reaction will not occur if the ΔG 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, ΔG = 0.d. The ΔG of a reaction depends only on the free energy difference between reactants and

products and is independent of how the reaction occurs.e. The ΔG 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 statei. S XΨP

b. Binding energy is the free energy released upon interaction of the enzyme and substratec. Binding energy is greatest when the enzyme interacts with the transition state, thus

facilitating the formation of the transition stated. Evidence for E-S Complex

i. Maximum velocity reached with constant concentration of enzymeii. 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 enzymec. Active sites create unique microenvironmentsd. The interactions of the enzyme and substrate at the active site involves multiple weak

interactionse. Enzyme specificity depends on the molecular architecture at the active site

7. Catalytic Mechanismsa. Binding – enzyme uses binding energy to increase rate of reactionb. The enzyme brings substrate together in an orientation that facilitates catalysis

i. Proximity and orientation (entropy reduction) ii. Electrostatic catalysis

iii. Preferential transition state bindingiv. 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 protonii. 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 Fita. 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 Enzymesa. Oxidoreductase catalyze oxidation-reduction reactions.

i. A– + B → A + B–

ii. Pi + glyceraldehyde-3-phosphate + NAD+ → NADH + H+ + 1,3-bisphosphoglycerate

b. Transferases move functional groups between molecules.i. A–X + B → A + B–X

c. Hydrolyases cleave bonds with the addition of water.i. A–B + H2O → A–OH + B–H

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-Rii. 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 moleculesi. Ab + C → A–C + b

ii. Ab + cD → A–D + b + c10. Enzyme Co-factors

a. Cofactorsi. Coenzymes

1. Co-substrates (weakly bound)2. Prosthetic groups (tightly bound)

ii. Essential metal ions1. Activator ions (weakly bound)2. Active site ions (tightly bound)

11. Kinetics is the study of reaction ratesa. 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 –> Pii. 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 Equationi. Rate of the reverse reaction, E + P -> ES is ignored

1. Valid because measuring initial velocities, when [P] is very low2. 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 ESd. Equations needed to know

i.

ii.

iii.

iv.e. Michaelis Constant

i.ii. Michaelis Constant

1. KM = [S] when V0 = ½ Vmaxf. 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 efficiency1. 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 reactionsi. 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 Inhibitorsi. Irreversible – bind got enzymes very tightly, often forming a covalent bond, and

inactivate themii. Reversible – interact more loosely with enzymes and can be displaced

1. Competitive2. Uncompetitive3. 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 Inhibitors1. Group-specific inhibitors

a. React with specific side chainb. Ex: DIPF

2. Affinity label or reactive substrate analogsa. Structurally similar to substrateb. Covalent binds to active sites

3. Suicide inhibitor or mechanism-based inhibitorsa. Modified substratesb. Bind as substratec. Catalyzed to reactive intermediate that inactivates the enzyme

thru covalent modification14. Irreversible Inhibitors Can Be Used to Map the Active Site

a. Acetylcholinesterase + DIPF -> Inactivate + F- + H+

b. Affinity label – reactivity analogc. Bromoacetol phosphate – an affinity label for triose phosphate isomerase (TPI)

i. Triose phosphate isomerase + bromoacetol phosphate -> Inactivate enzyme15. Suicide or mechanism-based inhibitor

a. MonoAmine Oxdiase Inhibitor (MAOIs) b. The suicide inhibitor removes E so that the [ES] is lower, Vmax is lower, and inhibition

cannot be overcome at high S0