Probing the Active Site of Tritrichomonas foetus Hypoxanthine‐Guanine‐Xanthine Phosphoribosyltransferase Using Covalent Modification of Cysteine Residues

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Abstract
The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) of Tritrichomonas foetus was inactivated by the thiol reagents iodoacetate and 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2). Iodoacetate inactivates the enzyme in a time-dependent and concentration-dependent manner that follows pseudo-first-order kinetics. However, the observation that total inactivation with iodoacetate was not achieved suggests that none of the reactive cysteine residues is directly involved in the catalytic activity of the enzyme. Nbs2 caused 50% inactivation rapidly, which was followed by gradual modifications of an additional three cysteine residues leading to complete enzyme inactivation. Analysis of the inactivation using the method developed by Tsou (1962) revealed that modification of two cysteine residues by Nbs2 is sufficient to impair the HGXPRTase activity. Tryptic digestion of HGXPRTase labeled with iodo[2-14C]acetic acid, followed by fractionation of the digest by HPLC and sequence analysis of the labeled peptides allowed the identification of Cys71, Cys129, Cys132, and Cys148 as the reactive cysteine residues. GMP and 5-phosphoribosyl-1-diphosphate provided complete protection against HGXPRTase inactivation by iodoacetate and against carboxymethylation of Cys129, Cys132, and Cys148, Cys71 was not protected by either substrate against iodoacetate, but its carboxymethylation caused no loss in enzyme activity either. There was also no substrate protection of Cys71 against Nbs2, which, however, caused 50% inactivation of the enzyme. Replacing the thionitrobenzoate (Nbs) moiety from Cys71 with cyanide resulted in a gradual recovery of the enzyme activity, which indicates that a steric hindrance at the active site was introduced by Nbs but removed by cyanide. Thus, our results demonstrate 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 Nbs2 hinders substrate binding which can, in turn, protect the cysteine residues from modifications. The substrate protection of Cys129 and Cys148 is probably also indicative of a conformational change in the protein structure brought about by substrate binding.