Human Hypoxanthine Phosphoribosyltransferase: I. Purification, Properties, and Specificity

Abstract

Hypoxanthine phosphoribosyltransferase (inosine monophosphate:pyrophosphate phosphoribosyltransferase EC 2.4.2.8) has been purified 50-fold from an acid-treated lysate of human erythrocytes. The average particle weight of the enzyme was estimated by gel filtration at 60,000. Between the pH values of 7.1 and 9.1 no marked difference in the initial velocity of IMP synthesis was observed. Kinetic studies of magnesium effects are consistent with the view that magnesium activates nucleotide synthesis by complexing with the substrate 5-phosphoribosyl 1-pyrophosphate. Guanine, hypoxanthine, and 6-mercaptopurine are good substrates for the enzyme, while xanthine and allopurinol are very poor substrates. No detectable nucleotides were formed from adenine, uric acid, uracil, azathioprine, and oxipurinol. The enzyme effectively binds purines with an oxo or thio group in position 6, but not those with a 6-amino group. A 2-amino group enhances binding whereas a 2-hydroxyl group diminishes it. Of the N-methyl purines, only the 1-methyl derivatives show significant binding to the enzyme. The important role of the imidazole ring of purine in binding is indicated by the relatively poor binding of the pyrazolo(3,4-d)pyrimidines and the v-triazolo(4,5-d)pyrimidines. That the imidazole portion itself is not sufficient for binding is shown by the high Ki values for 5-aminoimidazole-4-carboxamide and its formyl derivative.