A kinetic .alpha.-deuterium isotope effect for the binding of purine nucleosides to calf spleen purine nucleoside phosphorylase. Evidence for catalysis by distortion

Abstract

Kinetic ..cap alpha..-deuterium isotope effects, k/sub H//k/sub D/, for the phosphorylase catalyzed phosphorolysis of adenosine at pH 7.2 and inosine at pH 7.5, were determined by the competitive double labelling technique to be 1.047 +- 0.017 and 1.043 +- 0.004, respectively. From the magnitude of these values, the authors conclude that the vibrations associated with the ..cap alpha..-hydrogen have been loosened in going from the inbound reactant molecules to the transition state for binding. As the nucleoside is bound to the active site of the enzyme, distortion of the ribose ring has occurred, causing the 1'C to undergo a geometry change characteristic of partial sp/sup 3/ to sp/sup 2/ rehybrydization. The energy required to bring about this distortion is presumably supplied through some process by which the enzyme utilizes binding energy derived from favorable interactions with parts of the substrate other than the reacting group. The minimum energy needed to bring about this distortion was calculated to be 0.18 kcal/mole by application of the Streitweiser approximation.