Enzyme Stereospecificity

1
Enzyme Stereospecificity

• As a rule, enzymes demonstrate unerring
  stereospecificity in catalysis
• they distinguish between optical or geometrical
  pairs.
• they (almost) always use one form of an
  enantiomeric pair, unless their function is to
  interconvert isomers.
• they can distinguish between paired chemically
  like substituents in cases such as Caabc (CH2XY).
• Stereospecificity is observed because enzymes are
  asymmetric reagents comprised of L-amino acids.
• Interaction of two enantiomers (e.g. D- or L-
  phenylalanine methyl ester) with a symmetric
  reagent generates transition states that are
  enantiomeric and of equal energy.
• Interaction of these enantiomers with an enzyme
  generates diastereometric transition states
  having different reactivity, and will partition
  differently between reactants and products.

2
Enzyme Specificity

• Enzymatic catalysis always involves prior complex
  formation between the reactant and the enzyme
• The substrate binds to a specific region on the
  enzyme in every catalytic cycle, and catalysis
  only occurs at the active site.
• Specificity can be imposed on the binding step,
  on any subsequent catalytic step, or both.
• An enzyme oxidizing a-hydroxy acids to a-keto
  acids may bind only one enantiomer (binding
  specificity) or it may bind both but oxidize only
  one isomer.
• The active site of an enzyme consists of an array
  of amino acid functional groups (and in some
  cases coordinated metals) positioned in a
  specific three dimensional orientation.

3
Enzyme-Substrate Interactions

• The enzyme hexokinase catalyzes a phosphoryl
  transfer from ATP to C-6 of glucose
• Conformational changes in the tertiary
• structure of hexokinose resulting
• from its binding of glucose have
• been observed. This is called
• induced fit, where conformational
• changes in the flexible enzyme
• when bound to a substrate
• generates a transition state
• structure (enzyme-substrate)
• of relatively low energy.

4
Conceptual Models of Enzyme-Substrate Binding

• Fischer lock and key hypothesis
• Koshland induced-fit hypothesis
• Hypothesis involving strain or transition state
  stabilization

5
Model Reactions Strain and Distortion

• Inducing strain in the substrate and release of
  that strain in the transition state to products
  is known to increase the rate of many classes of
  reactions.
• A model reaction that illustrates this effect is
  the phosphate-ester hydrolysis of cyclic and
  acyclic analogues
• Under similar conditions the cyclic ester (I)
  hydrolyzes at a relative rate that is 108 times
  faster than the acyclic ester (II).
• Distortion and strain imposed by an enzyme on a
  bound substrate is expected to destabilize it
  relative to the transition state, thereby
  enhancing the rate of reaction.

6
Model Reactions Proximity Effects

• Enzyme literature makes reference to catalysis by
  approximation, referring to the capacity of
  enzymes to make reactants proximal - adjacent to
  each other.
• An increase in the effective concentration of
  reactants over that found in the bulk of the
  solution is expected to increase the rate of
  reaction
• Model compound studies demonstrate this effect by
  comparing rates for similar intra- versus
  intermolecular transformations.
• Imidazole-catalyzed hydrolysis of p-nitrophenyl
  acetate relates to an enzyme active site
  involving a histidine residue
• The bimolecular rate constant under a given set
• of conditions is kobs,1 35 min-1M-1 for this
  reaction.

7
Model Reactions Proximity Effects

• To assess the acceleration derived from
  covalently imposed proximity, the intramolecular
  analogues of this reaction were studied.
• The first-order rate
• constant for this
• unimolecular case
• under the same
• conditions was
• kobs,2 200 min-1.
• An effective molarity of imidazole in this
  intramolecular reaction can be calculated through
  a comparison of the rate constants
• kobs,2 kobs,1 Imidazoleeff
  or Imidazoleeff 5.7 M
• Inclusion of a third methylene group
• to permit a 6-member transition
• state enhances the rate further,
• increasing Imidazoleeff to 23.9 M.

8
Enzymes Transition State Theory

• Our treatment of closed catalytic reaction cycles
  usually includes the assignment of a rate
  determining step. Assuming this step is
  elementary, we can discuss its kinetics in terms
  of transition state theory.
• Depending on the reaction mechanism, catalysis
  influences reaction rates by.
• 1. increasing the concentration of activated
  complex
• Acid/base catalyzed organic transformations
• 2. providing an alternate reaction pathway
  requiring a lower energy transition state
• Organotransition metal catalyzed isomerization of
  olefins
• 3. Destabilizing the substrate through
  complexation
• Enzyme catalyzed organic transformations

9
Kinetics of Enzyme Catalyzed Reactions

• In spite of the apparent complexity of enzyme
  catalyzed processes, their reaction kinetics can
  be described by a relatively simple sequence of
  reactions which, while not elementary, are
  sufficient to account for the observed behaviour.
• Michaelis and Menten (1913)
• If we assign the second reaction as irreversible
  and rate determining,
• where,
• r reaction rate (mole l-1 s-1)
• S concentration of substrate (mole l-1)
• ET total enzyme concentration (mole l-1)
• Ke Equilibrium constant (l mole-1)

10
Kinetics of Enzyme Catalyzed Reactions

• Michaelis-Menten rate expressions are usually
  expressed in an alternate form
• where,
• Vmax k2ET (s-1)
• S concentration of substrate (mole l-1)
• Km Michaelis constant (mole l-1) 1/Ke
• Suppose an enzyme concentration of 510-7 mole
  /litre can support a reaction rate of,
• As we vary the concentration of substrate from
  0.001 M to 1.0 M, how does the ratio of ES /
  E respond?

11
Kinetics of Enzyme Catalyzed Reactions

• It is clear that a relatively small substrate
  concentration will bind a very high fraction of
  enzyme.
• Even more impressive is the activity of this
  enzyme-substrate
• complex, which allows just 0.55 mmoles/litre to
  support a reaction velocity of 310-5 s-1.