Adenine Phosphoribosyltransferase from Monkey Liver

The structural requirements for the binding of purines to adenine phosphoribosyltransferase from monkey liver were explored by the determination of the K; 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 hydroxylaminoand hydrazinopurines bound well; the binding of alkylaminopurines was moderately good only for small alkyl groups. With the exception of purine-6aldoxime, purine, and 6-methylpurine, other 6-substituted purines showed Ki values >10w3 M. All Z-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-4carboxamide reacted at rates which indicated a high maximal velocity. Conversely, 4-aminopyrazolo[3,4dlpyrimidine 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 (K, 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 I-pyrophosphate (low3 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 phosphoribosyltransf er 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. Mammalian tissues contain two distinct purine phosphoribosyltransferases, one for adenine (adenine phosphoribosyltransferase -adenosine monophosphate: pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) and another for hypoxanthine and guanine (hypoxanthine-guanine phosphoribosyltransferaseinosine monophosphate: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) (l-3). Previous studies (4, 5) have dealt with the substrate-binding specificity of the hypoxanthine-guanine enzyme. This report is concerned principally with the structural requirements for binding to the adenine enzyme in a preparation from rhesus monkey liver. Kinetic studies with hypoxanthine-guanine phosphoribosyltransferase suggested that magnesium ions activate IMP synthesis by forming a complex with 5.phosphoribosyl I-pyrophosphate (4). The considerably higher affinity of PP-ribose-Pl for the adenine enzyme compared with the hypoxanthine enzyme afforded an opportunity to test this view further.