Hydration active site

The approach taken above estimates the effect of the metal by simply considering its electrostatic effect (subjected, of course, to the correct steric constraint as dictated by the metal van der Waals parameters). To examine the validity of this approach for other systems let s consider the reaction of the enzyme carbonic anhydrase, whose active site is shown in Fig. 8.6. The reaction of this enzyme involves the hydration of C02, which can be described as (Ref. 5)... 

V. Helms and R. C. Wade, Hydration energy landscape of the active site cavity in... 

The detailed mechanism of inhibition of TEM-2 (class A) enzyme with clavulanate has been established (Scheme 1) [23,24], The inhibition is a consequence of the instability of the acyl enzyme formed between the /1-lactam of clavulanate and the active site Ser-70 of the enzyme. In competition with deacylation, the clavulanate acyl-enzyme complex A undergoes an intramolecular fragmentation. This fragmentation initially provides the new acyl enzyme species B, which is at once capable of further reaction, including tautomeriza-tion to an entity C that is much less chemically reactive to deacylation. This species C then undergoes decarboxylation to give another key intermediate enamine D, which is in equilibrium with imine E. The imine E either forms stable cross-linked vinyl ether F, by interacting with Ser-130 or is converted to the hydrated aldehyde G to complete the inactivation. 

It is believed that these effects derive from salt influences on some process (associated with hydration) involved in establishing activation free energy values for enzymes, although not an active site phenomenon. 

The crystal structure of the cobalt-substituted enzyme was obtained with bicarbonate bound to the metal (Iverson et al. 2000). The structure shows Asn 202 and Gln75 hydrogen bonded to the metal-bound bicarbonate, suggestzing potential roles for these residues in either transition-state stabilization or orientation and polarization of CO2 for attack from the zinc-hydroxyl (Fig. 11.5). The crystal structure also shows three discrete conformations for Glu 84, suggesting a role for this residue in the transfer of protons out of the active site indeed, kinetic analyses of Glu 84 variants combined with chemical rescue experiments establish this residue as critical for proton transfer (Tripp and Ferry 2000). The location of Glu 62 adjacent to Glu 84 suggests a potential role in proton transfer as well. Although kinetic analyses of site-specific variants establish an essential role for Glu 62 in the CO2 hydration steps (Eqs. 11.3 and 11.4), the results were inconclusive regarding an additional role in proton transfer (Eqs. 11.5 and 11.6). 

Surface catalysis affects the kinetics of the process as well. Saltzman et al. (1974) note that in the case of Ca -kaolinite, parathion decomposition proceeds in two stages with different first-order rates (Fig. 16.14). In the first stage, parathion molecules specifically adsorbed on the saturating cation are quickly hydrolyzed by contact with the dissociated hydration water molecules. In the second stage, parathion molecules that might have been initially bound to the clay surface by different mechanisms are very slowly hydrolyzed, as they reach active sites with a proper orientation. 

An example is the hydration of CO2, as catalyzed by carbonic anhydrasek The catalytic reaction requires proton transfer from the zinc-bound water at the active site to solution to regenerate Zn-OH in each catalytic cycle. The most efficient isozyme forms use His-64 as a nearby proton shuttle group other forms contain residues that are less effective in proton transfer and limit overall catalytic efficiency. 

There may be two proton transfers in the carbonic anhydrase II-catalyzed mechanism of CO2 hydration that are important in catalysis, and both of these transfers are affected by the active-site zinc ion. The first (intramolecular) proton transfer may actually be a tautomerization between the intermediate and product forms of the bicarbonate anion (Fig. 28). This is believed to be a necessary step in the carbonic anhydrase II mechanism, due to a consideration of the reverse reaction. The cou-lombic attraction between bicarbonate and zinc is optimal when both oxygens of the delocalized anion face zinc, that is, when the bicarbonate anion is oriented with syn stereochemistry toward zinc (this is analogous to a syn-oriented carboxylate-zinc interaction see Fig. 28a). This energetically favorable interaction probably dominates the initial recognition of bicarbonate, but the tautomerization of zinc-bound bicarbonate is subsequently required for turnover in the reverse reaction (Fig. 28b). 

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