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This study examined recombinant wild-type human phosphoribosylpyrophosphate synthetase 1 (wt-PRS1, EC 2.7.6.1) and the point mutant Asn114Ser PRS1 (N114S-Mutant) in cells of a patient with primary gout. Dynamic light-scattering and sedimentation velocity experiments indicated that the monomeric wt-PRS1 in solution was assembled into hexamers after adding the substrate ATP. However, this ATP-induced aggregation effect was not observed with N114S-Mutant, which has a 50% higher enzymatic activity than that of wt-PRS1. Synchrotron radiation circular dichroism spectroscopy revealed that the point mutation causes an increase of alpha-helix content and a decrease of turn content. Examination of the crystal structure of wt-PRS1 indicated that 12 hydrogen bonds formed by 6 pairs of N114 and D139 have an important role in stabilizing the hexamer. We suggest that the substitution of S114 for N114 in N114S-Mutant leads to the rupture of 12 hydrogen bonds and breakage of the PO43- allosteric site where PO43- functions as a fixer of the ATP-binding loop. Therefore, we consider that formation of the hexamer as the structural basis of the ADP allosteric inhibition is greatly weakened by the N114S mutation, and that alteration of the ATP-binding loop conformation is the key factor in the increased activity of N114S-Mutant. These two factors could be responsible for the high level of activity of N114S-Mutant in this patient.

Summary The antiherpetic agent 9-β-D-arabinofuranosyladenine (araA) needs to be phosphorylated to its 5′-triphosphate to be effective as an inhibitor of herpes simplex virus replication. Adenosine kinase and deoxycytidine kinase are assumed to convert araA to its 5′-monophosphate. We now found that araAMP is converted to its 5′-triphosphate through a direct pyrophosphate transfer from 5-phosphoribosyl-1-pyrophosphate (PRPP) by PRPP synthetase. The efficiency of phosphorylation of araAMP to araATP is about 5% of that of AMP, as estimated from their Vmax/Km ratios for PRPP synthetase. AraATP is converted to araAMP by PRPP synthetase at a 4-fold higher Km but similar Vmax as ATP.

Quinolinate phosphoribosyl transferase was rapidly inactivated in Escherichia coli exposed to hyperbaric oxygen. The enzyme is essential for de novo biosynthesis of NAD in E. coli and man. Because of its sensitivity and essentiality, inactivation of this enzyme is proposed as a significant mechanism of cellular oxygen toxicity. Niacin which enters the NAD biosynthetic pathway below the oxygen-poisoned enzyme provided significant protection against the decrease in pyridine nucleotides and the growth inhibition from hyperoxia in E. coli and could be useful in cases of human oxygen poisoning.

The effect of 2,3-diphosphoglycerate (2,3-DPG) upon three partially purified enzymes from human erythrocytes, 5-phosphoribosyl-l-pyrophosphate synthetase (PRibPP synthetase, E.C.2.7.6.1), IMP:pyrophosphate phosphoribosyltransferase (E.C.2.4.2.8 HPRibTase) and AMP:pyrophosphate phosphoribosyltransferase (E.C.2.4.2.7 APRibTase) has been studied kinetically. PRibPP synthetase was not greatly inhibited at physiologic concentrations of 2,3-DPG at optimal phosphate concentration, but it was inhibited appreciably by 2,3-DPG at physiologic phosphate levels. Both HPRibTase and APRibTase were inhibited by 2,3-DPG at its normal intracellular concentration. 2,3-DPG is a competitive inhibitor with respect to the substrates, ribose-5-phosphate and 5-phosphoribosyl-l-pyrophosphate. The Ki values for these inhibitions are given and the possible physiologic significance of these findings are discussed.

Summary The regulation of phosphoribosylpyrophosphate synthetase purified from human peripheral blood lymphocytes has been studied. Enzyme activity was stimulated by inorganic phosphate, IMP and GMP and inhibited by ADP and, to a lesser extent, by AMP. The results suggest that phosphoribosylpyrophosphate synthetase may play an important role in the mechanism of lymphocyte proliferation.

An increased activity of phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate is demonstrated in extracts of skin fibroblast cultures derived from a patient with Lesch-Nyhan syndrome. This eccessive response of the phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate results in increased levels of phosphoribosylpyrophosphate and thus contributes to purine overproduction characteristic of this disorder. The level of enzyme response in skin fibroblast extracts from the carrier mother was between activity of the patient and normals, further suggesting the x-linkage of human phosphoribosylpyrophosphate synthetase.