Enzymatic Synthesis of Phosphoribosylamine in Human Cells

Abstract Two enzyme activities which catalyze phosphoribosyl-1-amine synthesis from phosphoribosyl pyrophosphate and glutamine and from phosphoribosyl pyrophosphate and ammonia were partially purified from human lymphoblasts (Burkitt lymphoma) maintained in tissue culture. The two enzyme activities were separated from each other by Sephadex G-100 chromatography. The elution pattern from a Sephadex G-100 column showed two distinct peaks of enzyme activities: peak activity for glutamine phosphoribosyl pyrophosphate amidotransferase corresponded to a molecular weight of approximately 95,000; peak activity of the ammonia-requiring enzyme activity corresponded to a molecular weight of 67,000. The lower molecular weight fraction could represent a separate enzyme activity, or it could be a subunit of glutamine phosphoribosyl pyrophosphate amidotransferase, which uses ammonia preferentially. Both enzyme activities required either Mg2+ or Mn2+ for catalytic activity, but optimal Mn2+ concentration for the ammonia-dependent activity was 0.25 mm. Concentrations above 0.25 mm inhibited ammonia-dependent catalytic activity, while optimal Mn2+ concentration for glutamine phosphoribosyl pyrophosphate amidotransferase was 2 mm. KCl (50 to 100 mm) inhibited phosphoribosyl-1-amine synthesis from ammonia and phosphoribosyl pyrophosphate, but did not inhibit glutamine phosphoribosyl pyrophosphate amidotransferase. Glutamine phosphoribosyl pyrophosphate amidotransferase was more heat-labile (t½ = 4 min) than was the lower molecular weight polypeptide which used ammonia for synthesis. The catalytic activity of the enzyme fraction which catalyzes phosphoribosyl-1-amine from ammonia and phosphoribosyl pyrophosphate was not inhibited by glutamine. The apparent Km values were 0.15 mm for phosphoribosyl pyrophosphate and 2 mm for ammonia. The Km values for glutamine phosphoribosyl pyrophosphate amidotransferase were 0.1 mm for phosphoribosyl pyrophosphate and 1 mm for glutamine. Both enzyme activities were sensitive to inhibition by adenosine 5'-monophosphate, guanosine 5'-monophosphate, and allopurinol ribonucleotide. The effect of ammonia on the early steps of de novo purine biosynthesis was investigated in the intact human lymphoblast maintained in tissue culture. Addition of 10 mm NH4Cl (at pH 7.5) stimulated the rate of purine biosynthesis in the intact cell of 168%, while the addition of adenine or hypoxanthine to the incubation medium inhibited the rate of the early steps of purine biosynthesis. Ammonia stimulation of α-N-formylglycinamide ribonucleotide synthesis was not mediated by an increase in cellular glutamine. Preincubation of cells with 1 mm 6-diazo-5-oxo-l-norleucine for 30 min inhibited glutamine-dependent α-N-formylglycinamide ribonucleotide synthesis by 80%, but reduced ammonia-dependent α-N-formylglycinamide ribonucleotide synthesis by only 20% (unpublished observations). These cells had no detectable glutamine synthetase activity, nor was glutamine synthesis detected following the addition of ammonia to the incubation medium (unpublished observations). The addition of 0.5 µm adenine inhibited glutaminedependent α-N-formylglycinamide ribonucleotide synthesis by 44%, while the addition of 2 µm hypoxanthine inhibited this synthesis by 42%. Ammonia-dependent α-N-formylglycinamide ribonucleotide synthesis was inhibited by 33% by 0.5 µm adenine and by 47% by 2 µm hypoxanthine. These observations indicate that ammonia-dependent phosphoribosyl-1-amine may contribute significantly to the regulation of the rate of de novo purine biosynthesis in human tissues. The regulation of purine biosynthesis is of particular interest in gout and in malignant diseases.