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

Abstract

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 m M ⁻¹ min ⁻¹ and the K _i was 2.7 ± 0.7 m M . 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 reactivemore » 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.« less

Authors:

Terzyan, Simon S. ^[1]; Cook, Paul F. ^[2]; Heroux, Annie ^[3]; Hanigan, Marie H. ^[1]

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+ Show Author Affiliations

1. Univ. of Oklahoma Health Sciences Center, Oklahoma City, OK (United States)
2. Univ. of Oklahoma, Norman, OK (United States)
3. Brookhaven National Lab. (BNL), Upton, NY (United States)

Publication Date:

Wed Apr 05 00:00:00 EDT 2017

Research Org.:

Brookhaven National Laboratory (BNL), Upton, NY (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Basic Energy Sciences (BES)

OSTI Identifier:

1435158

Alternate Identifier(s):

OSTI ID: 1401773

Report Number(s):

BNL-203489-2018-JAAM
Journal ID: ISSN 0961-8368

Grant/Contract Number:  

SC0012704

Resource Type:

Accepted Manuscript

Journal Name:

Protein Science

Additional Journal Information:

Journal Volume: 26; Journal Issue: 6; Journal ID: ISSN 0961-8368

Publisher:

The Protein Society

Country of Publication:

United States

Language:

English

Subject:

36 MATERIALS SCIENCE; enzyme inactivation; enzyme kinetics; crystal structure; protein conformation; human gamma-glutamyl transpeptidase; gamma-glutamyl transferase