IDENTIFICATION OF THE ACTIVE-SITES OF HUMAN AND SCHISTOSOMAL HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASES BY GMP-2',3'-DIALDEHYDE AFFINITY LABELING

Labeling of human and schistosomal hypoxanthine-guanine phosphoribosyl transferases (HGPRTases) with GMP-2',3/-dialdehyde (ox-GMP) results in nearly complete inactivation of the enzymes. Digestion of the [H-3]ox -GMP-modified HGPRTases with trypsin followed by high-performance liqu id chromatographic fractionation, partial amino acid sequencing, and m ass spectral analysis of the labeled peptides revealed that four pepti des from each of the two HGPRTases were labeled with ox-GMP. The concl usion from these studies indicates that two segments of the human enzy me protein, Ser 4-Arg 47 and Ser 91-Arg 100, and one region in the sch istosomal enzyme, Gly 95-Lys 133, were labeled by ox-CMP. Since the ox -GMP labeling of human HGPRTase was effectively blocked by either GMP or PRibPP, whereas that of schistosomal HGPRTase was inhibited only by GMP [Kanaaneh, J., Craig, S. P., III, & Wang, C. C. (1994) fur. J. Bi ochem. 223, 595-601], the two labeled peptides in human enzyme may be involved in binding to both GMP and PRibPP while the one peptide in sc histosomal enzyme may be implicated only in GMP binding. We have also confirmed a previous observation [Keough, D. T., Emmerson, B. T., & de Jersey, J. (1991) Biochim. Biophys. Acta 1096, 95-100] that carboxyme thylation of Cys 22 in the human HGPRTase by iodoacetate was inhibited by PRibPP. We also demonstrated that the carboxymethylation of Cys 25 in schistosomal HGPRTase by iodoacetate was specifically blocked by P RibPP. Apparently, the N-terminal regions in both enzymes are involved in PRibPP binding. The fact that ox-GMP labels the N-terminal region in human enzyme but not in schistosomal enzyme and that PRibPP protect s against ox-GMP labeling in human enzyme but not in schistosomal enzy me both suggest that the amino-terminal PRibPP-binding site may be in close proximity to the GMP-binding site in human HGPRTase but not in s chistosomal HGPRTase. This clear distinction between the active sites of human and schistosomal HGPRTases could be further exploited for pot ential opportunities for antischistosomal chemotherapy.