Human phosphoribosylpyrophosphate synthetase. Comparison of purified normal and mutant enzymes

Abstract

Human phosphoribosylpyrophosphate synthetase has been purified 4500-fold to electrophoretic homogeneity from the erythrocytes of normal individuals and of two brothers in whom excessive activity of this enzyme results in excessive rated of purine nucleotide and uric acid synthesis de novo and gouty arthritis. Structural differences between the normal and mutant enzymes are indicated by a lower isoelectric point for the mutant enzyme (pI 4.85) than for the normal enzyme (pI 5.10); decreased electrophoretic mobility of the mutant preparation on cellulose acetate gel at low inorganic phosphate concentrations; increased (2.4-fold) inactivation of the mutant enzyme activity relative to the normal by identical amounts of a specific antiserum which precipitates identical quantities of normal and mutant enzyme; increased thermal lability of the mutant enzyme at 55 degrees; and an increased (2.2-fold) specific enzyme activity for the mutant enzyme despite the comparable purity of the preparations. Antibody inactivation, quantitative precipitin, and immunodiffusion studies as well as the disparity in specific enzyme activities during the course of purification suggest that a structural alteration in the mutant enzyme leads to increased catalytic activity per enzyme molecule, either from a primary alteration in the structural gene(s) for phosphoribosylpyrophosphate synthetase or from a post-transcriptional alteration in the enzyme. Purified preparations of normal and mutant enzymes showed nearly identical affinity constants for magnesium and the substrates, ATP and ribose 5-phosphate, as well as similar inhibition constants for the products, PP-ribose-P and AMP, and the inhibitors ADP, GDP, and 2,3-diphosphoglycerate. An increased maximal velocity of the reaction was, thus, the sole kinetic difference identified. The increased velocity of the mutant enzyme reaction was constant over a range of inorganic phosphate concentrations from 0.1 to 100 mM. Subunit molecular weights of the enzyme preparations, estimated by sodium dodecyl sulfate-polyacrylamide electrophoresis, were identical (32,000), although the undenatured mutant enzyme showed a greater proportion of stainable protein in the smaller of two molecular weight forms (both greater than 500,000) of the enzyme demonstrated on polyacrylamide gel electrophoresis in the presence of 1 mM sodium phosphate.