Kinetic alpha-deuterium isotope effects for Escherichia coli purine nucleoside phosphorylase-catalyzed phosphorolysis of adenosine and inosine.

Kinetic alpha-deuterium isotope effects have been measured for the purine nucleoside phosphorylase-catalyzed phosphorolysis of adenosine and inosine by a competitive double label technique at saturating concentrations of the second substrate, phosphate. Under these conditions the observed isotope effect, kH/kD, is on the second order rate constant, Vmax/Km, for reaction of nucleoside with the Michaelis complex of enzyme and phosphate. For adenosine, at neutral pH, the isotope effect is unity. For inosine, kH/kD was determined as a function of pH (the numbers in parentheses are the ratios of Vmax at that pH to Vmax at pH 7.3): 1.10 at pH 5.0 (0.19); 1.10 at pH 6.1 (0.72); 1.01 at pH 7.3 (1.00); 1.16 at pH 8.4 (0.22); and 1.18 at pH 9.4 (0.04). These values suggest a mechanism for purine nucleoside phosphorylase involving a change in rate-limiting step from one at pH values near neutrality for which cleavage of the nucleoside C-N bond is not rate limiting to a step at extremes of pH with a transition state having considerable oxocarbonium ion-like character.