Variant human phosphoribosylpyrophosphate synthetase altered in regulatory and catalytic functions.

Abstract

A B S T R A C T An inherited, structurally abnormal and superactive form of the enzyme 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) synthetase (EC 2.7.6.1) has been characterized in fibroblasts cultured from a 14-yr-old male (S.M.) with clinical manifestations of uric acid overproduction present since infancy. PPribose-P synthetase from the cells of this child showed four-to fivefold greater than normal resistance to purine nucleotide (ADP and GDP) feedback inhibition ofenzyme activity and hyperbolic rather than sigmoidal inorganic phosphate (Pi) and by an accelerated, age-related decrement in enzyme activity in lysates of erythrocytes separated by specific density. Despite the diminished amount of PP-ribose-P synthetase in the S.M. erythrocyte population, S.M. erythrocytes had increased PPribose-P concentration and increased rates of incorporation of ['4C]adenine and hypoxanthine into acid-soluble nucleotides during incubation at 1 mM Pi. These findings provided further confirmation of the extent to which PP-ribose-P synthesis is modulated in the normal cell at physiological Pi concentration by purine nucleotide inhibition of PP-ribose-P synthetase.The activity and kinetic characteristics of PPribose-P synthetase from fibroblasts of the mother of patient S.M. indicated that this woman was a heterozygous carrier of the enzyme defect expressed in hemizygous manner by her son. INTRODUCTIONThe high-energy sugar phosphate 5-phosphoribosyl I-pyrophosphate (PP-ribose-P)' is an intermediate in the synthesis of purine, pyrimidine and pyridine nucleotides. The synthesis of PP-ribose-P from ATP and ribose-5-phosphate (Rib-5-P) is catalyzed by the enzyme PP-ribose-P synthetase (E.C. 2.7.6.1) in a reaction requiring Mg2+ and inorganic phosphate (Pi). Studies of PP-ribose-P production by intact cells (1, 2) and analyses of the kinetic characteristics of purified microbial (3-5) and mammalian (6-10) PP-ribose-P synthetases indicate that a substantial number of effector compounds including substrates, inhibitors, activators, and products influence enzyme activity.

prsB is an allele of the Salmonella typhimurium prsA gene: characterization of a mutant phosphoribosylpyrophosphate synthetase

“…A temperature-sensitive E. coli PRPP synthetase mutation, prs-2 (9), also maps to this general region of the prs-100 mutation (lla). A mutant human PRPP synthetase, which has properties reminiscent of prs-100, is also known (2). This mutant has an elevated maximal velocity, is reduced in sensitivity to allosteric inhibitors, and is thermally labile (2).…”

“…A mutant human PRPP synthetase, which has properties reminiscent of prs-100, is also known (2). This mutant has an elevated maximal velocity, is reduced in sensitivity to allosteric inhibitors, and is thermally labile (2). It will be of interest to learn whether these E. coli and human mutants are altered in the same region of the PRPP synthetase as identified for the S. typhimurium prs-100 mutant enzyme.…”

Selective expression of phosphoribosylpyrophosphate synthetase superactivity in human lymphoblast lines.

“…Superactivity of 5-phosphoribosyl 1-pyrophosphate (PRPP)' synthetase (EC 2.7.6.1), the enzyme catalyzing synthesis of the purine regulatory intermediate PRPP from ATP and ribose-5-phosphate (ribose-5-P) (1), is an inherited disorder in which excessive enzyme activity is associated with uric acid overproduction and gout (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Erythrocytes and cultured fibroblasts from affected individuals show increases in PRPP concentration and generation as well as excessive rates of PRPP-dependent purine synthetic reactions (4)(5)(6)(7)(8)(13)(14)(15).…”

“…Erythrocytes and cultured fibroblasts from affected individuals show increases in PRPP concentration and generation as well as excessive rates of PRPP-dependent purine synthetic reactions (4)(5)(6)(7)(8)(13)(14)(15). Increased rates of purine synthesis de novo have been shown both in affected males in vivo (2-4, 6-8, 10, 11, 16) and in their fibroblasts in vitro (3-5, 7, 11, 14, 15).…”

“…Among the heterogeneous kinetic defects underlying PRPP synthetase superactivity are: (a) catalytic defects due to increased maximal reaction velocity (6-8, 11, 18); (b) regulatory defects due to diminished responsiveness to purine nucleotide inhibitors of enzyme activity ( 14,19); (c) substrate binding defects resulting in increased affinity for ribose-5-P (4); and (d) combined catalytic and regulatory defects (5). Studies of superactive enzymes in each of these classes (4-8, 14, 18, 19) have provided evidence for structural alteration underlying superactivity.…”