Another example is the carboxypeptidase hydrolysis of peptides. Carboxypeptidase is a metalloenzyme, containing a Zn⁺⁺ at the active site.

Carboxypeptidase (1yme)	Carboxypeptidase, Closeup of Catalytic Site

• The Zn is the orange atom in the center, bound to histidines 69 and 196 and to Glu72

• These ligands serve simply to bind the Zn, and do not play an active role in catalysis

• Arg145 and Glu270 are the catalytic residues

The Zn provides electrophilic catalysis, becoming bonded to the carbonyl oxygen.

• The ester produced by this step would then undergo reaction with a water molecule by the same process to give net cleavage of the peptide bond.

• Alternatively, the Glu-270 functions as a base, facilitating the attack of a water, and thus forming the products in a single step:

If the latter mechanism is correct, this example is base catalysis by the Glu, rather than nucleophilic catalysis.

At present, the evidence seems to favor base catalysis [Mock, in Comprehensive Biological Catalysis, Vol. 1, Academic Press, 1998; p. 425; Guo et al., J. Am. Chem. Soc., 2009, 131, 9780].

Transition State Stabilization:

Rate of reaction is determined by the activation energy - the difference in energy between the reactants and the transition state.

• Hence, one way to increase rate is to increase the energy of the reactants (red line)

Once upon a time, when biochemistry was younger, many persons believed that the lowering of the activation enthalpy observed in enzymatic catalysis arose from strain induced upon binding of enzyme and substrate.

• That is, binding stretched the bonds in the substrate so that they were partially broken before reaction began.

• However, the evidence is clear that the energies required for partial bond breaking are much greater than the binding energies of substrates

However, the alternative to raising the energy of the substrate to obtain a lower activation enthalpy is to lower the energy of the transition state (green line).

• The demonstration of inhibition of enzyme activity by transition state analogs is strong evidence for the operation of transition state stabilization.

One example: adenosine deaminase, which catalyzes the reaction:

If the enzyme is presented with a substrate analog lacking the amino group

• The enzyme binds it, and adds the molecule of water at the 6-position

• However, the resulting structure, a transition state analog, is bound much more tightly than either the natural substrate or the substrate analog

• The enzyme is thereby inactivated

The K_i cited is essentially the inverse of the Michaelis constant, and is called the inhibition constant.

The smaller the value, the greater the inhibition. Observation of this inhibition led to the design of the antibiotic coformycin, which is itself a transition state analog.

Adenosine Deaminase with Coformycin Bound (1a4l)	Adenosine Deaminase with Deazapurine Bound (1a4m)

Note the active site as a "hole", with the inhibitor bound at the bottom of it.