title: gamma-Glutamylcyclotransferase: inhibition by D-beta-aminoglutaryl-L-alanine and analysis of the solvent kinetic isotope effect.
abstract: Spin-echo NMR spectroscopy was used to record the cleavage of a gamma-glutamyl--amino-acid by (5-L-glutamyl)-L-amino-acid 5-glutamyltransferase (cyclizing) (gamma-glutamylcyclotransferase) in human erythrocyte hemolysates. The Michaelis-Menten steady-state kinetic parameters were obtained by fitting the integrated Michaelis-Menten equation to the reaction time curves. The product, L-5-oxoproline, was shown to be an inhibitor of the reaction. The active site of the enzyme was probed by studies of the inhibition by D- and L-beta-aminoglutaryl-L-alanine which are the beta-amino-acid isomers of D- and L-gamma-glutamyl-L-alanine (the latter being a natural substrate of the enzyme); the D-isomer was the more potent inhibitor (Ki = 0.30 +/- 0.02 mmol/l water). When the alanyl alpha-carboxyl of the inhibitor was reduced to a hydroxyl (i.e. to give D-beta-aminoglutaryl-L-alaninol) the potency of inhibition was reduced. The previously reported kinetic isotope effect of solvent 2H2O on the enzyme-catalyzed reaction has been further studied using a proton inventory. We propose that the solvent kinetic isotope effect is due to an intramolecular proton transfer between the glutamyl amino group and the peptide bond nitrogen.
authors.affiliation: Department of Biochemistry, University of Sydney, Australia.
authors.forename: M J
authors.initials: MJ
authors.lastname: York
authors.forename: M J
authors.initials: MJ
authors.lastname: Crossley
authors.forename: S J
authors.initials: SJ
authors.lastname: Hyslop
authors.forename: M L
authors.initials: ML
authors.lastname: Fisher
authors.forename: P W
authors.initials: PW
authors.lastname: Kuchel
mesh_terms: D000217:Acyltransferases / Q000097:blood*; D001665:Binding Sites; D004151:Dipeptides / Q000494:pharmacology*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D012997:Solvents; D005722:gamma-Glutamylcyclotransferase / Q000037:antagonists & inhibitors / Q000097:blood*
chemical_list: D004151:Dipeptides; D012997:Solvents; C061156:beta-aminoglutarylalanine; D000217:Acyltransferases; D005722:gamma-Glutamylcyclotransferase