Substrate inhibition in a human variant of hypoxanthine-guanine phosphoribosyltransferase.
Hypoxanthine-guanine phosphoribosyltransferase from a young man with purine overproduction and decreased purine salvage in fibroblast cultures was found to have low activity at concentrations of purine substrates at which the enzyme from normal individuals showed near maximal activity. The low enzyme activity was not associated with changes in the values of the Km(app) and Vmax(app) for any of the enzyme substrates. However, the enzyme activity was susceptible to substrate inhibition by hypoxanthine and guanine. The values obtained for the true Km, true Vmax, and true Ki for hypoxanthine were 26 +/- 10 microM, 1761 +/- 382 microunits/mg of protein, and 80 +/- 20 microM, respectively. The pattern of the substrate inhibition, as seen on a plot of 1/v versus hypoxanthine concentration, was characteristic of that associated with the formation of a dead-end complex between the inhibitory substrate and an enzyme form with which it normally does not react. The nature of this enzyme form and that of the dead-end complex was determined from double inhibition experiments, which indicated that hypoxanthine interacted with an enzyme-PPi intermediate to form an enzyme-hypoxanthine-PPi dead-end complex. The trapping of the enzyme in this inactive form explains the low activity at high purine base concentrations. Further information as to the nature of the reaction mechanism was obtained from plots of the reciprocal of enzyme activity versus the reciprocal of PP-ribose-P concentration at different fixed hypoxanthine concentrations. A pattern characteristic of uncompetitive substrate inhibition was obtained. This is indicative of an ordered sequential binding of substrates on the enzyme; PP-ribose-P binding before hypoxanthine. Thus, the variant enzyme showed an ordered sequential reaction mechanism, with the inhibitory substrate forming a dead-end complex with an enzyme-PPi intermediate.
D006147:Guanine / Q000378:metabolism; D006801:Humans; D019271:Hypoxanthine; D007041:Hypoxanthine Phosphoribosyltransferase / Q000037:antagonists & inhibitors*; D007042:Hypoxanthines / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D010428:Pentosephosphates / Q000378:metabolism; D010754:Phosphoribosyl Pyrophosphate
D007042:Hypoxanthines; D010428:Pentosephosphates; D019271:Hypoxanthine; D006147:Guanine; D010754:Phosphoribosyl Pyrophosphate; D007041:Hypoxanthine Phosphoribosyltransferase