Localization of the 5-phospho-alpha-D-ribosyl-1-pyrophosphate binding site of human hypoxanthine-guanine phosphoribosyltransferase.
Human erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HPRT) is inactivated by iodoacetate in the absence, but not in the presence, of the substrate, 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRib-PP). Treatment of HPRT with [14C]iodoacetate followed by tryptic digestion, peptide separation and sequencing has shown that Cys-22 reacts with iodoacetate only in the absence of PRib-PP; this strongly suggests that Cys-22 is in or near the PRib-PP binding site. In contrast, Cys-105 reacts with [14C]iodoacetate both in the presence and absence of PRib-PP. Carboxymethylation of Cys-22 resulted in an increase in the Km for PRib-PP, but no change in Vmax. Storage of HPRT also resulted in an increase in the Km for PRib-PP and a decrease in its susceptibility to inactivation by iodoacetate. Dialysis of stored enzyme against 1 mM dithiothreitol resulted in a marked decrease in Km for PRib-PP. The stoichiometry of the reaction of [14C]iodoacetate with Cys-22 in HPRT leading to inactivation (approx. 1 residue modified per tetramer) showed that, in this preparation of HPRT purified from erythrocytes, only about 25% of the Cys-22 side chains were present as free and accessible thiols. Titration of thiol groups [with 5,5'-dithiobis(2-nitrobenzoic acid)] and the effect of dithiothreitol on Km for PRib-PP indicate that oxidation of thiol groups occurs on storage of HPRT, even in the presence of 1 mM beta-mercaptoethanol.
Keough
D T
DT
Department of Medicine, University of Queensland, Princess Alexandra Hospital, Woolloongabba, Australia.
Emmerson
B T
BT
de Jersey
J
J
D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D010446:Peptide Fragments / Q000737:chemistry; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism*
D007461:Iodoacetates; D010446:Peptide Fragments; D010754:Phosphoribosyl Pyrophosphate; D004228:Dithionitrobenzoic Acid; D007041:Hypoxanthine Phosphoribosyltransferase; D019807:Iodoacetic Acid