Purification and active site modification studies on glyoxalase I from monkey intestinal mucosa.
Glyoxalase I ((R)-S-lactoylglutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) from monkey intestinal mucosa was purified to homogeneity. The purified enzyme had a molecular weight of 48,000, composed of two apparently identical subunits. Active-site modification was carried out on the purified enzyme in presence and absence of S-hexylglutathione, a reversible competitive inhibitor of glyoxalase I. Modification by tetranitromethane and N-acetylimidazole caused inactivation of the enzyme. Inactivation by N-acetylimidazole was reversible with hydroxylamine treatment, suggesting the importance of tyrosine residues for the activity of the enzyme. The enzyme was inactivated by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, 2,4,6-trinitrobenzenesulphonic acid, pyridoxal phosphate and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, indicating the importance of tryptophan, lysine and glutamic acid/aspartic acid residues for the activity of the enzyme. The enzyme was inactivated by diethyl pyrocarbonate and the activity was not restored by hydroxylamine treatment, suggesting that histidine residues may not be important for activity. Modification by N-ethylmaleimide and p-hydroxymercuribenzoate did not affect its activity, indicating that sulphydryl groups may not be important for activity. These studies indicated that the amino acids present in the active site of glyoxalase I from intestinal mucosa which may be important for activity are tyrosine, tryptophan, lysine and glutamic acid/aspartic acid residues.
D000600:Amino Acids, Dicarboxylic / Q000502:physiology; D000818:Animals; D000838:Anions; D001059:Apoproteins / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D002412:Cations; D007413:Intestinal Mucosa / Q000201:enzymology*; D007791:Lactoylglutathione Lyase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008190:Lyases / Q000302:isolation & purification*; D008239:Lysine / Q000502:physiology; D008255:Macaca radiata; D008970:Molecular Weight; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014364:Tryptophan / Q000502:physiology; D014443:Tyrosine / Q000502:physiology
D000600:Amino Acids, Dicarboxylic; D000838:Anions; D001059:Apoproteins; D002412:Cations; D013438:Sulfhydryl Compounds; D014443:Tyrosine; D011732:Pyridoxal Phosphate; D014364:Tryptophan; D008190:Lyases; D007791:Lactoylglutathione Lyase; D008239:Lysine