Human Hypoxanthine Phosphoribosyltransferase

At an optional magnesium sulfate concentration (5 mm), initial velocity analyses of hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) purified from human erythrocytes resulted in parallel lines on double reciprocal plots. A nonparallel pattern was observed at 0.05 mm magnesium sulfate, but only at relatively high 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) concentrations. Product inhibition by mononucleotides was competitive with PP-ribose-P and noncompetitive with hypoxanthine. GMP, GDP, and GTP were more inhibitory than IMP. PPi was an effective inhibitor at low magnesium sulfate concentrations but an ineffective inhibitor at high levels of this salt. PPi stimulated exchange between IMP and 14C-hypoxanthine. This exchange was slow as compared with the rate of IMP synthesis, and pyrophosphorolysis was slow as compared with the exchange rate. Binding of 14C-GMP to the free enzyme was shown by Sephadex chromatography. The relative elution patterns of the radioactivity and the enzyme activity suggested that the bulk of the binding was noncovalent. The sulfhydryl reactants, p-chloromercuribenzoate and N-ethylmaleimide, inactivated the enzyme. Preliminary treatment with PP-ribose-P protected the enzyme against inactivation by these reactants. Preliminary treatment of the enzyme with PP-ribose-P also resulted in an initial burst of IMP synthesis when hypoxanthine was added. Since none of the previously considered mechanisms, ordered sequential and ping-pong, are entirely consistent with the experimental results, a mechanism involving alternate reaction sequences is considered.