Title: Human γ-glutamyl transpeptidase 1: Structures of the free enzyme, inhibitor-bound tetrahedral transition states, and glutamate-bound enzyme reveal novel movement within the active site during catalysis [Human gamma-glutamyl transpeptidase: Inhibitor binding and movement within the active site]

Abstract

γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within the active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement ofmore » the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. Lastly,tThese data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use.« less

Authors:

Terzyan, Simon S. ^[1]; Burgett, Anthony W. G. ^[2]; Heroux, Annie ^[3]; Smith, Clyde A. ^[4]; Mooers, Blaine H. M. ^[1]; 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)
4. SLAC National Accelerator Lab., Menlo Park, CA (United States)

Publication Date:

Tue May 26 00:00:00 EDT 2015

Research Org.:

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

Sponsoring Org.:

USDOE Office of Science (SC)

OSTI Identifier:

1341504

Report Number(s):

BNL-108431-2015-JA
Journal ID: ISSN 0021-9258; R&D Project: LS001

Grant/Contract Number:  

SC00112704

Resource Type:

Accepted Manuscript

Journal Name:

Journal of Biological Chemistry

Additional Journal Information:

Journal Volume: 290; Journal Issue: 28; Journal ID: ISSN 0021-9258

Publisher:

American Society for Biochemistry and Molecular Biology

Country of Publication:

United States

Language:

English

Subject:

36 MATERIALS SCIENCE; crystal structure; enzyme inactivation; human; oxyanion hole; protein conformation; N-terminal hydrolase; cysteine; gamma-glutamyl transferase; gamma-glutamyl transpeptidase; glutathione