Adenosine deaminase: viscosity studies and the mechanism of binding of substrate and of ground- and transition-state analog inhibitors

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Abstract
We have studied the effects of viscosogenic agents, sucrose and ficoll, on (1) the hydrolysis of adenosine and of 6-methoxypurine riboside catalyzed by adenosine deaminase and (2) the rates of association and dissociation of ground-state and transition-state analogue inhibitors. For adenosine, Vmax/Km is found to be inversely proportional to the relative viscosity with sucrose, an agent affecting the microscopic viscosity, while no effect is found with ficoll, an agent affecting the macroscopic viscosity. Viscosogenic agents have no effect on the kinetic constants for 6-methoxypurine riboside. Thus, the bimolecular rate constant, Vmax/Km = 11.2 .+-. 0.8 .mu.M-1 s-1, for the reaction with adenosine is found to be at the encounter-controlled limit while that for the reaction with the poor substrate 6-methoxypurine riboside, 0.040 .+-. 0.004 .mu.M-1 s-1, is limited by some other process. Viscosity-dependent processes do not make a significant (< 10%) contribution to Vmax. The dissociation constants for inhibitors are unaffected by visocity. The ground-state analogue inhibitor purine riboside appears to bind at a rate comparable to that of adenosine. However, the slower rates of association (0.16 - 2.5 .mu.M-1 s-1) and dissociation (5 .times. 10-6 to 12 s-1) of transition-state analogue inhibitors are affected by the viscosity of the medium to approximately the same extent as the encounter-controlled rates of association and dissociation of adenosine. A two-step mechanism, a weak prior-equilibrium binding step followed by a viscosity-dependent enzyme conformation change, seems to adequately account for both the low absolute magnitude and the viscosity dependence of the apparent second-order rate constans for transition-state analogue binding.

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