Inactivation of Tritrichomonas foetus and Schistosoma mansoni purine phosphoribosyltransferases by arginine-specific reagents.
The arginine-specific reagents phenylglyoxal and butane-2,3-dione irreversibly inactivate the Tritrichomonas foetus hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) and Schistosoma mansoni hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The inactivation of the tritrichomonal enzyme by phenylglyoxal follows time-dependent and concentration-dependent pseudo-first-order kinetics. Complete protection against inactivation is afforded by the addition of 25 microM GMP, whereas 5-phosphoribosyl-1-diphosphate (PRibPP) at 50-250 microM can only slow down the inactivation, without being protective. Digestion of [7-(14)C]phenylglyoxal-modified enzyme with trypsin and separation of the peptides by reverse-phase HPLC shows that only one radioactive peak is greatly diminished by incubation with 25 microM GMP or 1 mM PRibPP. Mass-spectral analysis identifies Arg155 as the target site of two molecules of phenylglyoxal that is protected by the substrates. This amino acid residue is positioned next to Tyr156, which is a highly conserved aromatic residue among all the purine phosphoribosyltransferases (PRT) and is always found stacked on top of the purine substrate. This may explain why phenylglyoxal labeling of Arg155 inactivates the enzyme and why GMP can protect Arg155 more effectively than PRibPP. Among the purine PRT in our possession, only schistosomal HGPRT, the only other enzyme that contains an arginine residue at the corresponding location (Arg187), was susceptible to phenylglyoxal and butane-2,3-dione. The presence of Lys185-Phe186 and Ser179-Trp180 at the corresponding locations in human HGPRT and Giardia lamblia GPRT, respectively, may explain their resistance to phenylglyoxal. Thus, Arg155 in T. foetus HGXPRT and Arg187 in S. mansoni HGPRT will be attractive targets for future studies.
Kanaani
J
J
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
Maltby
D
D
Somoza
J R
JR
Wang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D007202:Indicators and Reagents; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D010658:Phenylglyoxal / Q000494:pharmacology; D011487:Protein Conformation; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D016835:Tritrichomonas foetus / Q000201:enzymology* / Q000235:genetics
D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D001120:Arginine; D010430:Pentosyltransferases; C083867:hypoxanthine-guanine-xanthine phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D010658:Phenylglyoxal