Functional residues on the enzyme active site of glyoxalase I from bovine brain.
Bovine brain glyoxalase I was investigated in order to identify amino acid residues essential for its catalytic activity. This enzyme is a 44-kDa dimeric protein which exhibits a characteristic intrinsic fluorescence, with an emission peak centered at 342 nm. The total of eight tryptophan residues/molecule was estimated by using a fluorescence titration method. Low values of Stern Volmer quenching constants for the quenchers used indicated that the tryptophan residues are relatively buried in the native molecule. Similar results were obtained for glyoxalase I, purified from yeast and human erythrocytes. The activity of bovine brain glyoxalase I was found to be particularly sensitive to 2,3-butanedione and diethylpyrocarbonate, selective reagents for arginine and histidine residues, respectively. A minor effect was observed by treatment of the enzyme with other amino acid-specific reagents. A protective effect of the competitive inhibitor S-hexylglutathione was observed for all reagents used, indicating the presence of modified amino acids in or near the enzyme active site.
Lupidi
G
G
Department of Biology M.C.A., University of Camerino, Italy. lupidi@cambio.unicam.it
Bollettini
M
M
Venardi
G
G
Marmocchi
F
F
Rotilio
G
G
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D004047:Diethyl Pyrocarbonate / Q000378:metabolism / Q000494:pharmacology; D019281:Dimerization; D004852:Epoxy Compounds / Q000378:metabolism / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007791:Lactoylglutathione Lyase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D008970:Molecular Weight; D010658:Phenylglyoxal / Q000378:metabolism / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D017186:Titrimetry; D014364:Tryptophan / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology
D004852:Epoxy Compounds; D006639:Histidine; C007366:diepoxybutane; D014364:Tryptophan; D001120:Arginine; D007791:Lactoylglutathione Lyase; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal