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^-1 min^-1 and the K_i 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.

Keywords: crystal structure; enzyme inactivation; enzyme kinetics; gamma-glutamyl transferase; human gamma-glutamyl transpeptidase; protein conformation.

Figure 1

Chemical structures of a generic gamma‐glutamyl compound (left) and DON (right). Arrow indicates the gamma‐glutamyl bond in the generic gamma‐glutamyl compound.

Figure 2

Inhibition of hGGT1 by DON. (A) hGGT1 was preincubated at 37°C with 10 mM (closed circles), 5 mM (closed squares), 2.5 mM (closed triangles), 1.25 mM (inverted closed triangles) or 0 mM DON (open diamonds) for the time indicated. Immediately following the preincubation, the enzyme was assayed for activity. Data points are average of mean values for four experiments ± standard error. For some data points the error bars are smaller than the symbol. (B) The data for the inactivation of hGGT1 by DON were first‐order.

Figure 3

Crystal structure of DON‐bound hGGT1. A stereo ribbon presentation of the hGGT1 heterodimer with DON bound in the active site (5V4Q). The large subunit (chain A) is colored blue, and the small subunit (chain B) is colored green. Thr381, the N‐terminus of the small subunit, is shown as a green stick figure. The DON atoms are colored red (the diazo group of DON is released when DON binds to the enzyme and is not present in the final structure).

Figure 4

DON‐bound hGGT. (A) Stereo presentation of the model of DON with diazo nitrogen atoms removed fitted into initial F _o – F _c density map (contoured at 3σ level). (B) Final 2F _o – F _c map for DON molecule (contoured at 1.5σ level). Enzyme carbon atoms are colored yellow, DON carbon atoms are colored orange, oxygens are red, and nitrogens are blue.

Figure 5

DON‐bound hGGT. Schematic representation of interactions of DON in the active site of hGGT1. Enzyme carbon atoms are colored yellow, DON carbon atoms are colored orange, oxygens are red, and nitrogens are blue. The distances shown are in Angstroms.

Figure 6

Proposed mechanism of hGGT1 inactivation by DON. The amine of Thr381 activates the nucleophile by accepting a proton from the OG oxygen of Thr381 via OG atom of Thr399. The OG atom of Thr381 attacks the carbonyl carbon (C5) of the DON molecule (top left structure). These results in the formation of a tetrahedral adduct which is stabilized by the interaction between the carboxy‐oxygen of DON and the main chain nitrogen atoms of Gly473 and Gly474 (top right structure). An electron pair on N7 of DON migrates to generate an N‐N triple bond and a proton is transferred from the amine of Thr381 to the C6 of DON (bottom right structure). Attack of the α‐nitrogen of Thr381 on the C6 of DON cleaves the single C‐N bond of the DON molecule releasing N2 and results in the formation of a covalent bond between the α‐nitrogen of Thr381 and C6 of DON. The final product (detected in the x‐ray structure, Figs. 3–5) contains a six‐membered ring composed of N, CA, CB, OG of the Thr381 and the C5 and C6 atoms of DON (bottom left structure).