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.
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 of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. These 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.
authors.affiliation: From the Macromolecular Crystallography Laboratory, Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
authors.forename: Simon S
authors.initials: SS
authors.lastname: Terzyan
authors.affiliation: the Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019.
authors.forename: Anthony W G
authors.initials: AW
authors.lastname: Burgett
authors.affiliation: the Energy Sciences Directorate/Photon Science Division, Brookhaven National Laboratory, Upton, New York 11973.
authors.forename: Annie
authors.initials: A
authors.lastname: Heroux
authors.affiliation: the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025.
authors.forename: Clyde A
authors.initials: CA
authors.lastname: Smith
authors.affiliation: the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, and.
authors.forename: Blaine H M
authors.initials: BH
authors.lastname: Mooers
authors.affiliation: the Department of Cell Biology, University of Oklahoma Health Sciences Center, Stanton L. Young Biomedical Research Center, Oklahoma City, Oklahoma 73104 marie-hanigan@ouhsc.edu.
authors.forename: Marie H
authors.initials: MH
authors.lastname: Hanigan
mesh_terms: D000613:Aminobutyrates / Q000737:chemistry / Q000494:pharmacology; D001051:Apoenzymes / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D063065:Organophosphonates / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics
chemical_list: C569224:2-amino-4-((3-(carboxymethyl)phenyl)(methyl)phosphono)butanoic acid; D000613:Aminobutyrates; D001051:Apoenzymes; D004791:Enzyme Inhibitors; D063065:Organophosphonates; D011994:Recombinant Proteins; D018698:Glutamic Acid; D005723:gamma-Glutamyltransferase; C554782:gamma-glutamyltransferase, human
keywords: N-terminal hydrolase; crystal structure; cysteine; enzyme inactivation; gamma-glutamyl transferase; gamma-glutamyl transpeptidase; glutathione; human; oxyanion hole; protein conformation