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 phosphoribosyltransferases (HGPRTases) with GMP-2',3'-dialdehyde (ox-GMP) results in nearly complete inactivation of the enzymes. Digestion of the [3H]ox-GMP-modified HGPRTases with trypsin followed by high-performance liquid chromatographic fractionation, partial amino acid sequencing, and mass spectral analysis of the labeled peptides revealed that four peptides from each of the two HGPRTases were labeled with ox-GMP. The conclusion from these studies indicates that two segments of the human enzyme protein, Ser 4-Arg 47 and Ser 91-Arg 100, and one region in the schistosomal enzyme, Gly 95-Lys 133, were labeled by ox-GMP. 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) Eur. J. Biochem. 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 schistosomal 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 carboxymethylation 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 PRibPP. 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 protects against ox-GMP labeling in human enzyme but not in schistosomal enzyme 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 schistosomal HGPRTase. This clear distinction between the active sites of human and schistosomal HGPRTases could be further exploited for potential opportunities for antischistosomal chemotherapy.
Kanaani
J
J
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143, USA.
Maltby
D
D
Focia
P
P
Wang
C C
CC
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D007461:Iodoacetates / Q000378:metabolism; D019807:Iodoacetic Acid; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism / Q000494:pharmacology; D012550:Schistosoma mansoni / Q000201:enzymology*; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D007461:Iodoacetates; D010446:Peptide Fragments; C088765:guanosine 2',3'-dialdehyde 5'-phosphate; D010754:Phosphoribosyl Pyrophosphate; D006157:Guanosine Monophosphate; D001120:Arginine; D007041:Hypoxanthine Phosphoribosyltransferase; D014357:Trypsin; D003545:Cysteine; D019807:Iodoacetic Acid