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.

Keywords: N-terminal hydrolase; crystal structure; cysteine; enzyme inactivation; gamma-glutamyl transferase; gamma-glutamyl transpeptidase; glutathione; human; oxyanion hole; protein conformation.

FIGURE 1.

Chemical structure of γ-glutamyl substrates. In a GGT-catalyzed reaction, a nucleophile (yellow arrow) attacks the δ-carbon of the glutamate moiety, and the γ-glutamyl bond (green arrow) is cleaved. The nucleophile in hGGT1 is the side chain oxygen of Thr-381, which forms an acyl bond with the substrate in the enzyme-substrate complex.

FIGURE 2.

Apo-Structure of hGGT1. A, a stereo ribbon presentation of the apo-structure of hGGT1 (4Z9O). The large subunit is colored blue, and the small subunit is colored green. The N terminus of the large subunit (N_L) and the C terminus of the large (C_L) and small (C_S) subunits are marked. The N terminus of the small subunit is Thr-381, the nucleophile in the GGT-catalyzed reaction, and its stick model in red is shown within the active site (orange oval). B, two conformations of Thr-381 observed in the apo-structure of hGGT1 (4Z9O). The oxygens are colored red, nitrogens are colored blue, and the carbons are colored yellow for one orientation and gold for the other.

FIGURE 3.

Serine-borate bound hGGT1 (Protein Data Bank code 4ZC6). A, structure of the serine-borate complex. In the hGGT1-catalyzed reaction, the side chain oxygen of Thr-381 initiates a nucleophilic attack on the boron, which results in the formation of tetrahedral structure with a covalent bond between the oxygen of Thr-381 and the boron. B, stereo presentation of the model of serine-borate fitted into an initial F_o − F_c density map (contoured at 3σ level). Enzyme carbon atoms are colored yellow; serine-borate carbon atoms are colored orange, oxygens are red, nitrogens are blue, and boron is pink. The red sphere is a water molecule (W). C, LIGPLOT diagram of the interactions of the serine-borate complex with hGGT1. Atoms are colored as in B with the exception that the carbon atoms are colored black. The water molecule is cyan.

FIGURE 4.

GGsTop-bound hGGT1 (4ZBK). A, structure of GGsTop (2-amino-4-([3-(carboxymethyl) phenyl] (methyl)phosphono)-butanoic acid)). In the hGGT1-catalyzed reaction, the side chain oxygen of Thr-381 initiates a nucleophilic attack on the phosphate of GGsTop, which results in the formation of a covalent bond between the oxygen of Thr-381 and the phosphate of GGsTop and the cleavage of the phosphorus-oxygen bond of the GGsTop phenol releasing the phenol. B, stereo presentation of the model of cleaved GGsTop fitted into the 2F_o − F_c density map (contoured at the 1.5 σ level). Enzyme carbon atoms are colored yellow; GGsTop carbon atoms are colored orange, oxygens are red, nitrogens are blue, and phosphate is green. C, LIGPLOT diagram of the interactions between GGsTop and hGGT1. Atoms are colored as in B with the exception that the carbons are colored black. The water molecule is cyan. D, stereo view of the solvent-accessible surface of the active site of the GGsTop-bound hGGT1 with a model of cleaved GGsTop. Enzyme carbon atoms are colored white. The colors of the other atoms are the same as in B.

FIGURE 5.

Glutamate-bound hGGT1 (4ZCG). A, structure of glutamate, which is the second product of the GGT1 reaction. B, stereo presentation of the model of Glu fitted into an F_o − F_c density map calculated after initial rigid body refinement and contoured at 3σ level. Enzyme carbon atoms are colored yellow; glutamate carbon atoms are colored orange, oxygens are red, and nitrogens are blue. C, LIGPLOT diagram of the interactions between hGGT1 and a glutamate molecule in the active site of the enzyme. Colors of the atoms are the same as in A with the exception that the carbon atoms are black. The water molecule is colored cyan.

FIGURE 6.

Least squares superposition of CA atoms of apo-hGGT1 (4Z9O, red), serine-borate bound hGGT1 (4ZC6, green), and glutamate-bound hGGT1 (4ZCG, blue) structures. The numbers show the first and last residues of the corresponding structure: the lid loop (residues 428–439), the oxyanion loop (residues 470–475), the C-terminal part of helix α14 (residues 323–330), and the loop connecting β16 and β17 strands (residues 505–514).