Open Accessγ‐Glutamylcyclotransferase: inhibition by d ‐β‐aminoglutaryl‐ l ‐alanine and analysis of the solvent kinetic isotope effect
Author(s) -
YORK Michael J.,
CROSSLEY Maxwell J.,
HYSLOP Serena J.,
FISHER Megan L.,
KUCHEL Philip W.
Publication year - 1989
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1989.tb14994.x
Subject(s) - chemistry , kinetic isotope effect , alanine , stereochemistry , enzyme kinetics , amino acid , enzyme , substrate (aquarium) , solvent , peptide , active site , peptide bond , organic chemistry , biochemistry , deuterium , physics , oceanography , quantum mechanics , geology
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 ( K i = 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 2 H 2 O 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.