CATALYSIS IN HUMAN HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE - ASP-137 ACTS AS A GENERAL ACID BASE/

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyzes th e reversible formation of IMP and GMP from their respective bases hypo xanthine (Hx) and guanine (Gua) and the phosphoribosyl donor 5-phospho ribosyl-1-pyrophosphate (PRPP), The net formation and cleavage of the nucleosidic bond requires removal/addition of a proton at the purine m oiety, allowing enzymic catalysis to reduce the energy barrier associa ted with the reaction. The pH profile of k(cat) for IMP pyrophosphorol ysis revealed an essential acidic group with pK(a), of 7.9 whereas tho se for IMP or GMP formation indicated involvement of essential basic g roups, Based on the crystal structure of human HGPRTase, protonation/d eprotonation is likely to occur at N7 of the purine ring, and Lys 165 or Asp 137 are each candidates for the general base/acid. We have cons tructed, purified, and kinetically characterized two mutant HGPRTases to test this hypothesis, D137N displayed an 18-fold decrease in k(cat) for nucleotide formation with Hx as substrate, a 275-fold decrease in k(cat) with Gua, and a 500-fold decrease in k(cat) for IMP pyrophosph orolysis. D137N also showed lower K-D values for nucleotides and PRPP, The pH profiles of k(cat) for D137N were severely altered. In contras t to D137N, the k(cat) for K165Q was decreased only 2-fold in the forw ard reaction ansi was slightly increased in the reverse reaction. The K-m and K-D values showed that K165Q interacts with substrates more we akly than does the wild-type enzyme. Pre-steady-state experiments with K165Q indicated that the phosphoribosyl transfer step was fast in the forward reaction, as observed with the wild type, In contrast, D137N showed slower phosphoribosyl transfer chemistry, although guanine (300 0-fold reduction) was affected much more than hypoxanthine (32-fold re duction), In conclusion, Asp137 acts as a general catalytic acid/base for HGPRTase and Lys165 makes ground-state interactions with substrate s.