P. Shih et R. Wolfenden, ENZYME-SUBSTRATE COMPLEXES OF ADENOSINE AND CYTIDINE DEAMINASES - ABSENCE OF ACCUMULATION OF WATER ADDUCTS, Biochemistry, 35(15), 1996, pp. 4697-4703
Adenosine deaminase has been reported to bind the product inosine (the
substrate for the reverse reaction) as inosine 1,6-hydrate, considere
d similar in structure to the transition state for adenosine deaminati
on (Wilson & Quiocho, 1994), Accumulation on the enzyme of inosine 1,6
-hydrate would be surprising, because this compound is an actual inter
mediate, probably approaching the transition state, in oxygen exchange
between water and the C=O group of inosine, a reaction previously sho
wn 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 deox
yinosine with C-13, expecting an upfield shift of 70-110 ppm if signif
icant rehybridization to sp(3) had occurred at the carbonyl group. Ins
tead, the results show a very small shift (similar to 1.3 ppm), indica
ting 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 t
o retain its sp(2) hybridization at C-4, after binding by Escherichia
coli cytidine deaminase, an enzyme that catalyzes O-18 exchange from w
ater into uridine. These findings are consistent with the general expe
ctation, based on the unfavorable equilibrium of activation of enzyme-
bound substrates, that enzymes should not accumulate appreciable conce
ntrations of intermediates whose free energies approach that of the tr
ansition state in substrate transformation.