γ‐Glutamylcyclotransferase: inhibition by d‐β‐aminoglutaryl‐l‐alanine and analysis of the solvent kinetic isotope effect

Abstract

Spin‐echo NMR spectroscopy was used to record the cleavage of a γ‐glutamyl–amino‐acid by (5‐L‐glutamyl)‐L‐amino‐acid 5‐glutamyltransferase (cyclizing) (γ‐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‐β‐aminoglutaryl‐L‐alanine which are the β‐amino‐acid isomers of D‐ and L‐γ‐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 α‐carboxyl of the inhibitor was reduced to a hydroxyl (i.e. to give D‐β‐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. Copyright © 1989, Wiley Blackwell. All rights reserved