Regulation of Purine Ribonucleotide Synthesis by End Product Inhibition

Abstract

1. Partially purified preparation of phosphoribosylpyrophosphate amidotransferase [EC 2.4.2.14] contaminated with a small amount of glutaminase was obtained from a purine requiring mutant of Bacillus subtilis cultured with a limiting supply of adenine. The enzyme was stabilized completely while storage in a freezer by the presence of both GSH (10 mM) and EDTA (2mM). Optimum pH was about 7.5. Apparent Michaelis constants for phosphoribosylpyrophosphate and glutamine were 8.66×10⁻⁵M and 4.15× 10⁻³M, respectively. 2. The enzyme was inhibited strongly by AMP and ADP, weakly by GMP, and partially by GTP, while other purine and pyrimidine nucleotides showed almost no effect. 3. Double reciprocal plots of the reaction rate against phosphoribosylpyrophosphate concentration curved upward in the presence of the above mentioned inhibitors while it was linear in the absence. These inhibitors were all competitive to phosphoribosylpyrophosphate. AMP and ADP were competitive also to glutamine but GTP and GMP were of mixed type. The rate-inhibitor concentration curves for AMP and ADP were S-shaped and 100% inhibition was obtained by increasing the concentration of the inhibitors. In contrast, the curves for GTP and GMP were hyperbolic and the inhibition by GTP was partial.