PROBING THE ACTIVE-SITE OF TRITRICHOMONAS-FETUS HYPOXANTHINE-GUANINE-XANTHINE PHOSPHORIBOSYLTRANSFERASE USING COVALENT MODIFICATION OF CYSTEINE RESIDUES

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase ) of Tritrichomonas foetus was inactivated by the thiol reagents iodoa cetate and 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs(2)). Iodoacetate i nactivates the enzyme in a time-dependent and concentration-dependent manner that follows pseudo-first-order kinetics. However, the observat ion that total inactivation with iodoacetate was not achieved suggests that none of the reactive cysteine residues is directly involved in t he catalytic activity of the enzyme. Nbs(2) caused 50% inactivation ra pidly, which was followed by gradual modifications of an additional th ree cysteine residues leading to complete enzyme inactivation. Analysi s of the inactivation using the method developed by Tsou (1962) reveal ed that modification of two cysteine residues by Nbs(2) is sufficient to impair the HGXPRTase activity, Tryptic digestion of HGXPRTase label ed with iodo[2-C-14]acetic acid, followed by fractionation of the dige st by HPLC and sequence analysis of the labeled peptides allowed the i dentification of Cys71, Cys129, Cys132, and Cys148 as the reactive cys teine residues. GMP and 5-phosphoribosyl-1-diphosphate provided comple te protection against HGXPRTase inactivation by iodoacetate and agains t carboxymethylation of Cys179, Cys132, and Cys148. Cys71 was not prot ected bg either substrate against iodoacetate, but its carboxymethylat ion caused no loss in enzyme activity either. There was also no substr ate protection of Cys71 against Nbs(2), which, however, caused 50% ina ctivation of the enzyme. Replacing tire thionitrobenzoate (Nbs) moiety from Cys71 with cyanide resulted in a gradual recovery or the enzyme activity which indicates that a steric hindrance at the active site wa s Introduced by Nbs but removed by cyanide. Thus, our results demonstr ate that although the reactive cysteine residues in HGXPRTase are not directly involved in the catalytic activity, modification of cysteine residues 129, 132, and 148 by iodoacetate or Nbs(2) hinders substrate binding which can, in turn, protect the cysteine residues from modific ations. The substrate protection of Cys129 and Cys148 is probably also indicative of a conformational change in the protein structure brough t about by substrate binding.