INHIBITION OF HUMAN 5-PHOSPHORIBOSYL-1-PYROPHOSPHATE SYNTHETASE BY YLAMINO)PYUIMIDO[5,4-D]PYRIMIDINE-5'-MONOPHOSPHATE - EVIDENCE FOR INTERACTION AT THE ADP ALLOSTERIC SITE

The kinetics of inhibition by the aminopyrimidopyrimidine nucleotide o -8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyri midine-5'-monophosph ate (APP-MP) were assessed with two human isozymes of 5-phosphoribosyl -1-pyrophosphate synthetase (PRS) (PRS1 and PRS2) and a mutant enzyme, S.M. PRS1, derived from an individual with PRS hyperactivity. In the presence of 1 mM potassium phosphate, APP-MP inhibited PRS1 and PRS2 w ith half-maximal inhibition (IC50) at 5.2 mu M and 23.8 mu M, respecti vely. The degree of inhibition for both enzymes was highly dependent o n the phosphate concentration; IC50 values were 70 times higher in the presence of 50 mM potassium phosphate. APP-MP exhibited mixed noncomp etitive-uncompetitive inhibition against PRS1, with a K-il value of 6. 1 mu M and a K-is value of 14.6 mu M, and produced parabolic secondary plots of slope or intercept versus APP-MP concentration. In compariso n, inhibition of PRS1 by ADP was of a mixed noncompetitive-competitive type, with a K-il value of 9.6 mu M and a K-is value of 2.8 mu M. A s imilar kinetic analysis was completed using S.M. PRS1, a mutant enzyme with a single amino acid substitution resulting in diminished sensiti vity to feedback inhibition by nucleotides. The noncompetitive compone nt of ADP inhibition of PRS1 was absent with S.M. PRS1 and ADP inhibit ion was purely competitive, with a K-i of 6.4 mu M. APP-MP was a very poor inhibitor of S.M. PRS1, displaying uncompetitive characteristics and a K-i of 1.6 mM. These data indicate that APP-MP inhibits PRS1 wit h a strong element of noncompetitive inhibition and appears to interac t specifically at the allosteric site used by ADP. These results contr ast with those obtained with ADP, which has a strong component of ATP competitive inhibition and binds at the ATP site as well as at a secon d, allosteric, site.