Studies on the nature of the regulation by purine nucleotides of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase from Ehrlich ascites-tumour cells

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Abstract
The progress curves of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase activity plotted against 5-phosphoribosyl pyrophosphate concentration were hyperbolic in nature. The inhibition of the former enzyme by AMP and GMP and of the latter enzyme by IMP [inosine monophosphate] and GMP showed completely competitive characteristics. The effect of temperature on the reaction of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase was examined. The energy of activation of tne former enzyme decreased at temperatures greater than 27 degrees and that of the latter enzyme at temperatures greater than 23 degrees. For each enzyme, the change in the heat of formation of the 5-phosphoribosyl pyrophosphate-enzyme complex at the critical temperature was approximately equal to the change in the energy of activation but was in the opposite direction. The inhibitor constants with both enzymes in the presence of nucleotides varied in different ways with temperature from the Michaelis constants for 5-phosphoribosyl pyrophosphate indicating that different functional groups were involved in binding substrates and inhibitors. ATP was found to stimulate adenine-phosphoribosyltransferase activity at concentrations less than about 250 [mu] [image] and to inhibit the enzyme at concentrations greater than 250 [mu] [image]. The stimulation was unaffected by 5-phosphoribosyl pyrophosphate concentration but the inhibitory effect could be overcome by increasing concentrations of this compound. At low concentrations ATP reversed the inhibition of adenine phosphoribosyltransferase by AMP and GMP to an extent dependent on their concentration. The properties of adenine phosphoribosyltransferase changed markedly on purification. Crude extracts of ascites-tumour cells had Michaelis constants for 5-phosphoribosyl pyrophosphate and adenine 75 and six times as high respectively as those obtained with purified enzyme. ATP had no stimulatory effect on activity of the purified enzyme or on that of crude extracts heated 15 min. or longer at 55 degrees. It is suggested that at low concentrations ATP is bound to an ''activator'' site which is separate from the substrate binding site of adenine phosphorytrans-ferase and that at high concentrations ATP competes with 5-phosphoribosyl pyrophosphate at the active site of the enzyme.