In the presence of excess magnesium, 5-phosphoribosyl pyrophosphate synthetase from Ehrlich ascites tumor cells showed hyperbolic responses to increasing concentrations of adenosine triphosphate and ribose 5-phosphate; concentrations required for half-maximal rates were 0.06 and 0.05 mM, respectively. The reaction had an absolute dependence upon phosphate and half-maximal rates were obtained with a concentration of 3.3 mM. Adenosine triphosphate, deoxyadenosine triphosphate, and O-adenylyl methylenediphosphonate were substrates for 5-phosphoribosyl pyrophosphate synthetase; no reaction was obtained with guanosine triphosphate, deoxyguanosine triphosphate, thymidine triphosphate, cytidine triphosphate, deoxycytidine triphosphate, uridine triphosphate, xanthosine triphosphate, or inosine triphosphate. Partial inhibitions of 5-phosphoribosyl pyrophosphate synthetase were obtained with adenosine monophosphate, guanosine monophosphate, and inosine monophosphate. Adenosine monophosphate was a mixed competitive and noncompetitive inhibitor with respect to MgATP2-; guanosine monophosphate was a noncompetitive inhibitor. Adenosine diphosphate was a competitive inhibitor with respect to adenosine triphosphate (Ki = 0.035 mM). The extracts used were contaminated with nucleoside diphosphokinase and other diphosphates were tested using O-adenylyl methylenediphosphonate as substrate; relatively weak inhibitions were obtained with the diphosphate of guanosine, cytidine, and uridine. Partial inhibitions of 5-phosphoribosyl pyrophosphate synthetase were obtained with several nucleoside triphosphates. In assays containing 1 mM ribose 5-phosphate, 0.5 mM MgATP2-, and 12.5 mM MgCl2, half-maximal inhibitions were obtained with 0.25 mM cytidine triphosphate, 0.7 mM deoxycytidine triphosphate, 0.2 mM guanosine triphosphate, 0.2 mM deoxyguanosine triphosphate, 0.4 mM thymidine triphosphate, 0.4 mM inosine triphosphate, 0.4 mM xanthosine triphosphate, and 0.4 mM uridine triphosphate; maximum inhibitions obtained were 84, 48, 52, 62, 78, 66, 75, and 75%, respectively. The inhibitions by thymidine triphosphate, cytidine triphosphate, and uridine triphosphate were competitive with respect to MgATP2-. Combinations of saturating levels of triphosphates gave inhibitions in between those obtained when the nucleotides were tested alone. © 1969, American Chemical Society. All rights reserved.
