Structure of 6‐diazo‐5‐oxo‐norleucine‐bound human gamma‐glutamyl transpeptidase 1, a novel mechanism of inactivation

Abstract

Intense efforts are underway to identify inhibitors of the enzyme gamma-glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma-glutamyl compounds and contributes to the pathology of asthma, reperfusion injury and cancer. The glutamate analog, 6-diazo-5-oxo-norleucine (DON), inhibits GGT1. DON also inhibits many essential glutamine metabolizing enzymes rendering it too toxic for use in the clinic as a GGT1 inhibitor. We investigated the molecular mechanism of human GGT1 (hGGT1) inhibition by DON to determine possible strategies for increasing its specificity for hGGT1. DON is an irreversible inhibitor of hGGT1. The second order rate constant of inactivation was 0.052 mM min and the K was 2.7 ± 0.7 mM. The crystal structure of DON-inactivated hGGT1 contained a molecule of DON without the diazo-nitrogen atoms in the active site. The overall structure of the hGGT1-DON complex resembled the structure of the apo-enzyme; however, shifts were detected in the loop forming the oxyanion hole and elements of the main chain that form the entrance to the active site. The structure of hGGT1-DON complex revealed two covalent bonds between the enzyme and inhibitor which were part of a six membered ring. The ring included the OG atom of Thr381, the reactive nucleophile of hGGT1 and the α-amine of Thr381. The structure of DON-bound hGGT1 has led to the discovery of a new mechanism of inactivation by DON that differs from its inactivation of other glutamine metabolizing enzymes, and insight into the activation of the catalytic nucleophile that initiates the hGGT1 reaction.

Sulfur-containing histidine compounds inhibit γ-glutamyl transpeptidase activity in human cancer cells

“…Enzyme assays were carried out using both human GGT (hGGT) isolated from membranes of human liver cancer cell line HepG2 or chronic B leukemic cells HG3 cells and the commercial equine kidney GGT (eqGGT; with a high percentage of identity with hGGT), maintaining fixed and saturating concentrations of ␥-glutamyl-para-nitroanilide (GpNa) and the acceptor glycyl-glycine (GlyGly) and varying the concentrations of the different testing compounds. Residual GGT activity in the presence of ovothiol A (ovo), isolated from P. lividus eggs in its disulfide form (41), was compared with that in the presence of the trimethyl-2-thiohistidine ergothioneine (erg) stabilized in thione form, the previously characterized GGT inhibitor DON (15), and dithiothreitol (DTT), used as a negative control (Fig. 2).…”

“…The most-known compounds that inhibit GGT include glutamine analogues and other amino acidic derivatives such as acivicin, 6-diazo-5-oxo-L-norleucine (DON), azaserine (15,16), sulfur derivatives of L-glutamic acid (17), and ␥-(monophenyl) phosphonoglutamate analogues (18,19). However, most of these compounds have proved to be toxic (20 -22) due to their interference with essential pathways such as the glutamate recycling involved in neurotransmission.…”

Probing the Interactions of Sulfur-Containing Histidine Compounds with Human Gamma-Glutamyl Transpeptidase

“…Here, we demonstrated that the des-methylated form of ovothiol (5-thio) also acts as a non-competitive-like inhibitor of GGT with Ki values very similar to ovo, and it is non-toxic towards human kidney embryonic cells. Moreover, we proved that both 5-thiohistidines, ovo and 5-thio, act as reversible GGT inhibitors compared to DON, which forms a stable and irreversible DON-GGT complex, consisting of a six-membered ring involving the catalytic Thr381 [31]. These data suggest that 5-thiohistidine derivatives can be finely modulated in in vitro and in vivo studies, thanks to their reversible mode of action.…”

“…GGT represents an important enzymatic target for the therapy of a wide variety of pathologies, such as tumors [22][23][24][25], liver fibrosis [17], ischemia/reperfusion-induced renal injury and asthma [26,27], thus the development of inhibitors of this enzyme with high specificity and low toxicity represents a booming field of research. Glutamine analogues have been developed, acting as irreversible competitive inhibitors due to their binding affinity for the donor site, but most of them have been abandoned in clinical trials due to toxicity [30][31][32]. Glutamic acid/glutamate derivatives have been proven to provide a valid therapeutic alternative, as these irreversible GGT inhibitors are more selective and less toxic [33][34][35][36][37].…”

“…The most well-known compounds that inhibit GGT include the glutamine analogues Acivicin ((2S)-2-amino-2-[(5S)-3-chloro-4,5-dihydro-1,2-oxazol-5-yl]acetic acid), DON (6-diazo-5-oxo-lnorleucine), and Azaserine (O-diazoacetyl-l-serine) [30,31]. However, most of these glutamine analogues have proved to be toxic, due to their inhibition of glutamine transferases and their interference with the glutamate-glutamine cycle, leading to innumerable side effects on pathways related to the recycling of the γ-aminobutyric acid neurotransmitter [32].…”