Enzyme-substrate complexes of adenosine and cytidine deaminases: absence of accumulation of water adducts.

Adenosine deaminase has been reported to bind the product inosine (the substrate for the reverse reaction) as inosine 1,6-hydrate considered similar in structure to the transition state for adenosine deamination (Wilson & Quiocho, 1994) Accumulation on the enzyme of inosine 1,6-hydrate would be surprising, because this compound is an actual intermediate, probably approaching the transition state, in oxygen exchange between water and the C==O group of inosine, a reaction previously shown to be catalyzed by adenosine deaminase (Wolfenden & Kirsch, 1968). The equilibrium constant for conversion of ES to ES*, in the oxygen exchange reaction, is less than 10-12. To investigate the structure of enzyme-bound inosine in a different way, we labeled deoxyinosine with 13C, excepting an upfield shift of 70-110 ppm if significant rehybridization to sp3 had occurred at the carbonyl group. Instead, the results show a very small shift (1.3 ppm), indicating that C-6 of 2'-deoxyinosine retains its sp2 hybridization after binding by calf intestinal adenosine deaminase. In a separate series of experiments, [4,5-13C]-2'-deoxyuridine was synthesized and found to retain its sp2 hybridization at C-4, after binding by Escherichia coli cytidine deaminase, an enzyme that catalyzes 18O exchange from water into uridine. These findings are consistent with the general expectation, based on the unfavorable equilibrium of activation of enzyme-bound substrates, that enzymes should not accumulate appreciable concentrations of intermediates whose free energies approach that of the transition state in substrate transformation.