Adenine Phosphoribosyltransferase from Monkey Liver SPECIFICITY AND PROPERTIES

Abstract The structural requirements for the binding of purines to adenine phosphoribosyltransferase from monkey liver were explored by the determination of the Ki values for 67 purines and purine analogues. Adenine was bound 40 times better than the most effectively bound analogues. Substitutions on the 6-amino group of adenine decreased binding to varying degrees. The hydroxylamino- and hydrazinopurines bound well; the binding of alkylaminopurines was moderately good only for small alkyl groups. With the exception of purine-6-aldoxime, purine, and 6-methylpurine, other 6-substituted purines showed Ki values g10-3 m. All 2-substituted derivatives of adenine tested were poorly bound, except 2-fluoroadenine. Ring nitrogen methylation of adenine markedly reduced binding. Adenine derivatives, substituted in position 8 with methyl, mercapto, or m-nitrophenyl, were effectively bound. Although poorly bound, 2,6-diaminopurine and 5-aminoimidazole-4-carboxamide reacted at rates which indicated a high maximal velocity. Conversely, 4-aminopyrazolo[3,4-d]pyrimidine was effectively bound but reacted slowly. Experiments with radioactive 5-aminoimidazole-4-carboxamide are consistent with earlier work which suggested that this substrate is converted to the nucleotide level by adenine phosphoribosyltransferase but not by the corresponding hypoxanthine enzyme. The kinetics of the magnesium activation of AMP synthesis was analogous to those previously reported for human hypoxanthine phosphoribosyltransferase. Although the apparent affinity (Km value = 0.037 mm) of adenine phosphoribosyltransferase for 5-phosphoribosyl 1-pyrophosphate was an order of magnitude greater than that of the human hypoxanthine enzyme, similar values for the dissociation constant of the dimagnesium salt of 5-phosphoribosyl 1-pyrophosphate (10-3 m) were estimated from the data obtained with both enzymes. This is consistent with the hypothesis that magnesium ions activate both these enzymatic reactions by forming a complex with the substrate, 5-phosphoribosyl 1-pyrophosphate. The adenine phosphoribosyltransfer activity of the monkey liver preparation was not decreased by sulfhydryl or serine modifiers, nor was it activated by sulfhydryl reagents; the residual phosphoribosyltransfer activity toward hypoxanthine was also insensitive to p-chloromercuribenzoate and dithiothreitol.