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 double dagger, 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 C-13, expecting an upfield shift of 70-110 ppm if significant rehybridization to sp(3) had occurred at the carbonyl group. Instead, the results show a very small shift (similar to 1.3 ppm), indicating that C-6 of 2'-deoxyinosine retains its sp(2) hybridization after binding by calf intestinal adenosine deaminase. In a separate series of experiments, [4,5-C-13]-2'-deoxyuridine was synthesized and found to retain its sp(2) hybridization at C-4, after binding by Escherichia coli cytidine deaminase, an enzyme that catalyzes O-18 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.