Inhibition of human 5-phosphoribosyl-1-pyrophosphate synthetase by 4-amino-8-(beta-D-ribofuranosylamino)-pyrimido[5,4-d]pyrimidine-5'- monophosphate: evidence for interaction at the ADP allosteric site.
The kinetics of inhibition by the aminopyrimidopyrimidine nucleotide 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine[-5' -monophosphate (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 with half-maximal inhibition (IC50) at 5.2 microM and 23.8 microM, respectively. The degree of inhibition for both enzymes was highly dependent on the phosphate concentration; IC50 values were 70 times higher in the presence of 50 mM potassium phosphate. APP-MP exhibited mixed noncompetitive-uncompetitive inhibition against PRS1, with a Kii value of 6.1 microM and a Kis value of 14.6 microM, and produced parabolic secondary plots of slope or intercept versus APP-MP concentration. In comparison, inhibition of PRS1 by ADP was of a mixed noncompetitive-competitive type, with a Kii value of 9.6 microM and a Kis value of 2.8 microM. A similar kinetic analysis was completed using S.M. PRS1, a mutant enzyme with a single amino acid substitution resulting in diminished sensitivity to feedback inhibition by nucleotides. The noncompetitive component of ADP inhibition of PRS1 was absent with S.M. PRS1 and ADP inhibition was purely competitive, with a Ki of 6.4 microM, APP-MP was a very poor inhibitor of S.M. PRS1, displaying uncompetitive characteristics and a Ki of 1.6 mM. These data indicate that APP-MP inhibits PRS1 with a strong element of noncompetitive inhibition and appears to interact specifically at the allosteric site used by ADP. These results contrast 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 second, allosteric, site.
Fry
D W
DW
Department of Cancer Research, Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan 48105, USA.
Becker
M A
MA
Switzer
R L
RL
D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology*; D000495:Allosteric Site; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D007700:Kinetics; D011742:Pyrimidine Nucleotides / Q000494:pharmacology*; D012268:Ribose-Phosphate Pyrophosphokinase / Q000037:antagonists & inhibitors*
D007527:Isoenzymes; D011742:Pyrimidine Nucleotides; C082815:4-amino-8-(ribofuranosylamino)pyrimido(5,4-d)pyrimidine-5'-phosphate; D000244:Adenosine Diphosphate; D012268:Ribose-Phosphate Pyrophosphokinase