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 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.

A Novel Prodrug of a γ‐Glutamylcyclotransferase Inhibitor Suppresses Cancer Cell Proliferation in vitro and Inhibits Tumor Growth in a Xenograft Mouse Model of Prostate Cancer

“…In previous studies, we reported on novel inhibitors of GGCT enzymatic activity. N ‐Glutaryl‐ l ‐alanine (GA) and other compounds inhibit GGCT activity in vitro, which can be measured using multistep reaction methods or with the originally developed GGCT probe, LISA‐101 . However, a major obstacle of using these candidate compounds as anticancer agents is that they are unable to penetrate living cell membranes efficiently.…”

Reconstruction of metabolic pathways for the cattle genome

“…Among 113 missing enzyme genes in the cattle metabolic pathways, the activities of six enzymes were reported in cattle (Table 4 ) [ 21 - 38 ]; 16 enzyme activities were reported in other mammals but not in cattle (Table 5 ) [ 39 - 67 ]. However, in both cases, corresponding mammalian genes have not been identified.…”

β-Amino acid isomers of a natural substrate of the enzyme γ-glutamyl-amino acid cyclotransferase. Synthesis of (3S)-3-aminoglutaryl-(S)-alanine and (3R)-3-aminoglutaryl-(S)-alanine

“…This paper reports the synthesis of (3S)-3-aminoglutaryl-(S)alanine (8), (3R)-3-aminoglutaryl-(S)-alanine (14), and (3R)-3aminoglutaryl-(S)-alaninol (22). These three dipeptides were required for structure-activity and inhibition studies on GCT.…”

“…Two routes to the dipeptides (8) and (14) have been investigated in this work. The preferred route is selective and makes use of an enzyme-catalysed cleavage of the prochiral diester (3) to the chiral half-ester (4) (Scheme 1).…”

“…Treatment of the diester (12) with trifluoroacetic acid gave the free acid peptide (13). Hydrogenolysis of compound (13) using 10% Pd-C as the catalyst gave the desired free peptide, (3R)-3-aminoglutaryl-(S)-alanine (14).…”