Human Phosphoribosylpyrophosphate Synthetase: Increased Enzyme Specific Activity in a Family with Gout and Excessive Purine Synthesis

Abstract

Among affected members of a family (B. family) with excessive purine production and gout, activity of phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) is 2.5- to 3.0-fold higher than among normal people. The molecular basis for this increased enzyme activity was studied. Antibody to purified human phosphoribosylpyrophosphate synthetase of erythrocytes was obtained from immunized rabbits. Studies with the IgG fraction of this antiserum show the presence of normal quantities of immunoreactive enzyme, but 2.5- to 3.0-fold higher activity per molecule in affected members of the B. family. In addition, by use of a stain for phosphoribosylpyrophosphate synthetase activity, a difference in electrophoretic mobility was demonstrated on cellulose acetate gel between the partially purified enzyme from normal people and an affected member of the B. family. These studies suggest that the enzyme aberration responsible for purine overproduction and gout in the B. family results from a structurally altered enzyme with increased activity per molecule.

Variant human phosphoribosylpyrophosphate synthetase altered in regulatory and catalytic functions.

“…Thus, S.M. PP-ribose-P synthetase resembles, in this respect, the superactive mutant enzyme with increased maximal velocity described by Becker et al, (10,18,19). In addition to possessing abnormal regulatory and catalytic properties, S.M.…”

“…Normal and SM fibroblast extracts were prepared by Sephadex G-25 chromatography and were equated for protein concentration. Each extract was then titrated with increasing quantities of IgG purified from the sera of an unimmunized rabbit and a rabbit immunized with highly purified normal erythrocte PPribose-P synthetase (19 synthetase was twofold greater than that of the normal enzyme even though, in parallel incubations, protein content of the immune precipitates resulting from addition of antibody to either of the extracts was identical for each ratio of antibody to enzyme. (Residual enzyme activities in the titration curves reflect activity of PP-ribose-P synthetase in the enzyme-antibody complex as previously demonstrated [25].)…”

“…This reduction in substrate concentrations was prompted by the observation that at Pi concentrations below 2.0 mM, considerable substrate inhibition of enzyme activity occurred in the presence of the usual substrate concentrations, 500 ,uM MgATP and 350 yM Rib-5-P. At the reduced substrate concentrations, substrate inhibition was not apparent, and the maximal velocities of the enzyme reaction at Thermal inactivation of partially purified fibroblast PPribose-P synthetase was studied at 54°C (10). Immunochemical inactivation of PP-ribose-P synthetase in fibroblast extracts prepared by Sephadex G-25 chromatography was investigated by titration of the extracts with the immunoglobulin (Ig)G fraction of monospecific rabbit antiserum to purified normal erythrocyte PP-ribose-P synthetase (19).…”

“…Study of inherited abnormalities of human PPribose-P synthetase, in which structurally variant forms of the enzyme with excessive activity lead to increased PP-ribose-P concentration and generation in conjunction with purine nucleotide overproduction and clinical gout (10,(14)(15)(16)(17)(18)(19)(20), have provided one line of evidence supporting a role for PP-ribose-P in the regulation of the rate of purine synthesis de novo (21)(22). Among these abnormal forms of the enzyme, superactive PP-ribose-P synthetases with either diminished sensitivity to feedback inhibition (16,17) or excessive reaction velocity per molecule of enzyme (10) have been described and extensively characterized.…”

Purine metabolism

“…An additional group of patients has now been defined in which the enzyme defect is not quite as severe as in children with the Lesch-Nyhan syndrome resulting in not only gouty arthritis and renal calculi, but also a variety of relatively mild attenuated neurologic disorders that include spinocerebellar syndrome, tlysartliria, spasticity, seizures, and mental retardation (3,4,23). An increased intracellular concentration of PP-ribose-P was also found in several families with gouty arthritis and normal activities of H P R T who nevertheless still showed a marked overproduction of uric acid (16)(17)(18)(24)(25)(26)(27)(28)(29)(30)(31)(32). Sperling's (24-27) and Becker's (16)(17)(18)(28)(29)(30)(31) identifications of a dominantly inherited increase i n specific activity of the enzyme PP-ribose-P synthetase as a cause of increased intracellular PP-ribose-P provided another different mutational cause for purine overproduction; thus two different types of mutations, each having in common a n increased intracellular concentration of PP-ribose-P, both produce as their end result an increased rate of purine biosynthesis.…”