Purine nucleoside phosphorylase. Catalytic mechanism and transition-state analysis of the arsenolysis reaction.

Purine nucleoside phosphorylase from calf spleen catalyzes the arsenolysis of inosine to form hypoxanthine and ribose 1-arsenate, which spontaneously hydrolyzes to ribose and arsenate. In the presence of H2(18)O, no 18O is incorporated into ribose, demonstrating that ribose 1-arsenate hydrolysis occurs by attack of water on the arsenic atom. Rapid reaction kinetics at 20 degrees C result in a biphasic rate curve with the first turnover occurring at a rate of 20 s-1 followed by a steady-state rate of 2 s-1. The product burst is consistent with rapid steps for substrate binding and arsenolysis followed by rate-limiting hypoxanthine release at a rate of 2 s-1. Purine nucleoside phosphorylase with bound [14C]inosine was mixed with excess unlabeled inosine and arsenate to determine relative rates for reaction or dissociation of bound inosine. The commitment factor (product formed/inosine released) was 0.19 at saturating arsenate, indicating that inosine binds to free enzyme and that bound inosine is not in thermodynamic equilibrium with free substrate. At neutral pH, kinetic isotope effects for the phosphorolysis reaction are small, indicating kinetic suppression. Kinetic isotope effects for arsenolysis were measured with [1'-3H]-, [2'-3H]-, [1'-14C]-, [9-15N]-, [4'-3H]-, and [5'-3H]inosine to provide experimental values of 1.118 +/- 0.003, 1.128 +/- 0.003, 1.022 +/- 0.005, 1.009 +/- 0.004, 1.007 +/- 0.003 and 1.028 +/- 0.004 respectively. Following correction for commitment factors, the intrinsic isotope effects were matched to a geometric transition-state model selected by bond-energy bond order vibrational analysis.(ABSTRACT TRUNCATED AT 250 WORDS)