Transition-state stabilization by adenosine deaminase: 1,6-addition of water to purine ribonucleoside, the enzyme's affinity for 6-hydroxy-1,6-dihydropurine ribonucleoside, and the effective concentration of substrate water at the active site

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Abstract
Positions of equilibria of highly unfavorable addition reactions, whose products are present at concentrations below the limits of detection, can be determined from equilibra of combination of anionic nucleophiles with quaternized enamies. Applied to the newly prepared 1-methylpurinium ribonucleoside cation, this method yields approximate equilibrium constants of 2 .times. 10-9 M-1 for addition of water and 4 .times. 10-5 M-1 for addition of N-acetylcysteine to neutral purine ribonucleoside, in dilute aqueous solution. Positions of 13C magnetic resonances and UV absorption and UV absorption maxima of the above complexes and comparison with those of adenosine deaminase complexes strongly suggest that purine ribonucleoside is bound by adenosine deaminase as the 1,6 covalent hydrate, not as a covalently bonded complex formed by addition of a thiol group at the active site. The favorable position of equilibrium of the hydration reaction on the enzyme, together with its extremely unfavorable position in free solution, indicates that the effective activity of substrate water at the active site is in the neighborhood of 1010 M. The Ki value of the acive diastereomer of 6-hydroxy-1,6-dihydropurine ribonucleoside is estimated as 1.6 .times. 1013 M, more than 8 orders of magnitude lower than the apparent dissociation constants of enzyme complexes with the substrate adenosine or the product inosine. The enzyme''s remarkable affinity for this hydrated species, which is vanishingly rare in free solution, seems understandable in terms of the hydrate''s close resemblance to a hydrated intermediate approaching the transition state is direct water attack on adensoine.