PURIFICATION AND PROPERTIES OF 5-PHOSPHORIBOSYL PYROPHOSPHATE AMIDOTRANSFERASE FROM ADENOCARCINOMA 755 CELLS

5-Phosphoribosyl 1-pyrophosphate amidotransferase has been partially purified from cultured Adenocarcinoma 755 cells by heat treatment, acid precipitation, and ammonium sulfate fractionation. Substrate saturation curves for 5-phosphoribosyl 1-pyrophosphate are sigmoid, and analysis of the data by the Hill equation shows an interaction coefficient of 1.9. The substrate concentration at half-maximal velocity, S0.5, is 0.47 mM. Saturation curves for the other substrate, L-glutamine, show no interaction for sites which bind this compound; but increasing amounts of 5-phosphoribosyl 1-pyrophosphate increase the binding of L-glutamine to the enzyme. 6-Diazo-5-oxo-L-norleucine is a competitive inhibitor of the binding of L-glutamine, with an inhibition constant, K1, of 12 μM. 2-Ethylamino-1,3,4-thiadiazole, a compound which apparently increases the rate of biosynthesis of nucleotides, does not stimulate 5-phosphoribosyl 1-pyrophosphate amidotransferase. All nucleotides tested inhibit the reaction to some extent. The most potent of all is 6-methylthiopurine ribonucleotide, for which a concentration of 90μM is required for 50% inhibition of the reaction, I0.5. Deoxyguanosine diphosphate, 6-thioguanylic acid, and 6-mercaptopurine ribonucleotide are also strong inhibitors; but inhibition by ribo- and deoxyribonucleoside triphosphates, except guanosine triphosphate, can be overcome at high magnesium ion concentration. Deoxyguanosine diphosphate and 6-thioguanylic acid display cooperativity between inhibitor binding sites, but the ribonucleotides of 6-mercaptopurine and 6-methylthiopurine do not. The presence of the nucleotide inhibitors does not alter the S0.5 value for 5-phosphoribosyl 1-pyrophosphate, and increased amounts of 5-phosphoribosyl 1-pyrophosphate do not alter the I0.5 values for the inhibitors. © 1969, American Chemical Society. All rights reserved.