Crystal structures of glutathione- and inhibitor-bound human GGT1: critical interactions within the cysteinylglycine binding site.

Overexpression of γ-glutamyl transpeptidase (GGT1) has been implicated in an array of human diseases including asthma, reperfusion injury, and cancer. Inhibitors are needed for therapy, but development of potent, specific inhibitors of GGT1 has been hampered by a lack of structural information regarding substrate binding and cleavage. To enhance our understanding of the molecular mechanism of substrate cleavage, we have solved the crystal structures of human GGT1 (hGGT1) with glutathione (a substrate) and a phosphate-glutathione analog (an irreversible inhibitor) bound in the active site. These are the first structures of any eukaryotic GGT with the cysteinylglycine region of the substrate-binding site occupied. These structures and the structure of apo-hGGT reveal movement of amino acid residues within the active site as the substrate binds. Asn-401 and Thr-381 each form hydrogen bonds with two atoms of GSH spanning the γ-glutamyl bond. Three different atoms of hGGT1 interact with the carboxyl oxygen of the cysteine of GSH. Interactions between the enzyme and substrate change as the substrate moves deeper into the active site cleft. The substrate reorients and a new hydrogen bond is formed between the substrate and the oxyanion hole. Thr-381 is locked into a single conformation as an acyl bond forms between the substrate and the enzyme. These data provide insight on a molecular level into the substrate specificity of hGGT1 and provide an explanation for seemingly disparate observations regarding the enzymatic activity of hGGT1 mutants. This knowledge will aid in the design of clinically useful hGGT1 inhibitors.

Sulfur-containing histidine compounds inhibit γ-glutamyl transpeptidase activity in human cancer cells.

γ-Glutamyl transpeptidase (GGT) is an enzyme located on the surface of cellular membranes and involved in GSH metabolism and maintenance of redox homeostasis. High GGT expression on tumor cells is associated with increased cell proliferation and resistance against chemotherapy. GGT inhibitors evaluated so far in clinical trials are too toxic for human use. In this study, using enzyme kinetics analyses, we demonstrate that ovothiols, 5(Nπ)-methyl thiohistidines of marine origin, act as noncompetitive inhibitors of GGT, with an apparent Ki of 21 µm, when we fixed the concentrations of the donor substrate. We found that these compounds are more potent than the known GGT inhibitor 6-diazo-5-oxo-l-norleucine and are not toxic toward human embryonic cells. In particular, cellular process-specific fluorescence-based assays revealed that ovothiols induce a mixed cell-death phenotype of apoptosis and autophagy in GGT-overexpressing cell lines, including human liver cancer and chronic B leukemic cells. The findings of our study provide the basis for further development of 5-thiohistidines as therapeutics for GGT-positive tumors and highlight that GGT inhibition is involved in autophagy.

Exogenous glutathione improves intracellular glutathione synthesis via the γ-glutamyl cycle in bovine zygotes and cleavage embryos.

Excess reactive oxygen species (ROS) generated in embryos during in vitro culture damage cellular macromolecules and embryo development. Glutathione (GSH) scavenges ROS and optimizes the culture system. However, how exogenous GSH influences intracellular GSH and improves the embryo developmental rate is poorly understood. In this study, GSH or GSX (a stable GSH isotope) was added to the culture media of bovine in vitro fertilization embryos for 7 days. The cleavage rate, blastocyst rate, and total cell number of blastocysts were calculated. Similarly to GSH, GSX increased the in vitro development rate and embryo quality. We measured intracellular ROS, GSX, and GSH for 0-32-hr postinsemination (hpi) in embryos (including zygotes at G1, S, and G2 phases and cleaved embryos) cultured in medium containing GSX. Intracellular ROS significantly decreased with increasing intracellular GSH in S-stage zygotes (18 hpi) and cleaved embryos (32 hpi). γ-Glutamyltranspeptidase ( GGT) and glutathione synthetase ( GSS) messenger RNA expression increased in zygotes (18 hpi) and cleaved embryos treated with GSH, consistent with the tendency of overall GSH content. GGT activity increased significantly in 18 hpi zygotes. GGT and GCL enzyme inhibition with acivicin and buthionine sulfoximine, respectively, decreased cleavage rate, blastocyst rate, total cell number, and GSH and GSX content. All results indicated that exogenous GSH affects intracellular GSH levels through the γ-glutamyl cycle and improves early embryo development, enhancing our understanding of the redox regulation effects and transport of GSH during embryo culture in vitro.

Involvement of γ-Glutamyl Transpeptidase in Ischemia/Reperfusion-Induced Cardiac Dysfunction in Isolated Rat Hearts.

GGsTop is a highly potent and specific, and irreversible γ-glutamyl transpeptidase (GGT) inhibitor without any influence on glutamine amidotransferases. The aim of the present study was to investigate the involvement of GGT in ischemia/reperfusion-induced cardiac dysfunction by assessing the effects of a treatment with GGsTop. Using a Langendorff apparatus, excised rat hearts underwent 40 min of global ischemia without irrigation and then 30 min of reperfusion. GGT activity was markedly increased in cardiac tissues exposed to ischemia, and was inhibited by the treatment with GGsTop. Exacerbation of cardiac functional parameters caused by ischemia and reperfusion, namely the reduction of left ventricular (LV) developed pressure and the maximum and negative minimum values of the first derivative of LV pressure, and the increment in LV end-diastolic pressure was significantly attenuated by GGsTop treatment. The treatment with GGsTop suppressed excessive norepinephrine release in the coronary perfusate, a marker for myocardial dysfunction, after ischemia/reperfusion. In addition, oxidative stress indicators in myocardium, including superoxide and malondialdehyde, after ischemia/reperfusion were significantly low in the presence of GGsTop. These observations demonstrate that enhanced GGT activity contributes to cardiac damage after myocardial ischemia/reperfusion, possibly via increased oxidative stress and subsequent norepinephrine overflow. GGT inhibitors have potential as a therapeutic strategy to prevent myocardial ischemia/reperfusion injury in vivo.

Impact of dapagliflozin, an SGLT2 inhibitor, on serum levels of soluble dipeptidyl peptidase-4 in patients with type 2 diabetes and non-alcoholic fatty liver disease.

AIMS: Soluble dipeptidyl peptidase-4 (sDPP-4) is secreted by hepatocytes and induces adipose tissue inflammation and insulin resistance. Sodium-glucose co-transporter-2 (SGLT2) inhibitors can improve hepatic steatosis by inhibiting hepatic de novo lipogenesis. We investigated the effects of dapagliflozin (an SGLT2 inhibitor) on serum levels of sDPP-4 in patients with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). METHODS: Fifty-seven patients with type 2 diabetes and NAFLD were randomized to a dapagliflozin group (5 mg/d for 24 weeks) (n = 33) or the control group (n = 24). Serum levels of sDPP-4 were measured with a commercial ELISA kit. Visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) areas were measured by dual bioelectrical impedance analysis. RESULTS: In a total of 57 patients, baseline serum sDPP-4 was positively correlated with aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transferase (GGT) and HOMA-IR Both VAT and SAT areas decreased significantly in the dapagliflozin group alone. Liver enzymes were decreased at 24 weeks in the dapagliflozin group, but were unchanged in the control group. Although both groups showed significant reduction of serum sDPP-4 after 24 weeks of treatment, the magnitude of decrease was significantly larger in the dapagliflozin group. Changes in liver enzymes during treatment with dapagliflozin were positively correlated with the change in serum sDPP-4, but not with changes in VAT volume or HbA1c. CONCLUSIONS: Improvement of liver dysfunction after treatment with dapagliflozin was associated with a decrease in serum sDPP-4, suggesting that reduction of serum sDPP-4 by SGLT2 inhibitors may be a therapeutic strategy for NAFLD/NASH in patients with type 2 diabetes that is independent of glucose lowering or weight loss.

Probing the Interactions of Sulfur-Containing Histidine Compounds with Human Gamma-Glutamyl Transpeptidase.

Gamma-glutamyl transpeptidase (GGT) is a cell surface enzyme involved in glutathione metabolism and maintenance of redox homeostasis. High expression of GGT on tumor cells is associated with an increase of cell proliferation and resistance against chemotherapy. GGT inhibitors that have been evaluated in clinical trials are too toxic for human use. We have previously identified ovothiols, 5(Nπ)-methyl-thiohistidines of marine origin, as non-competitive-like inhibitors of GGT that are more potent than the known GGT inhibitor, 6-diazo-5-oxo-l-norleucine (DON), and are not toxic for human embryonic cells. We extended these studies to the desmethylated form of ovothiol, 5-thiohistidine, and confirmed that this ovothiol derivative also acts as a non-competitive-like GGT inhibitor, with a potency comparable to ovothiol. We also found that both 5-thiohistidine derivatives act as reversible GGT inhibitors compared to the irreversible DON. Finally, we probed the interactions of 5-thiohistidines with GGT by docking analysis and compared them with the 2-thiohistidine ergothioneine, the physiological substrate glutathione, and the DON inhibitor. Overall, our results provide new insight for further development of 5-thiohistidine derivatives as therapeutics for GGT-positive tumors.

Targeted γ-secretase inhibition of Notch signaling activation in acute renal injury.

The Notch pathway has been reported to control tissue damage in acute kidney diseases. To investigate potential beneficial nephroprotective effects of targeting Notch, we developed chemically functionalized γ-secretase inhibitors (GSIs) targeting γ-glutamyltranspeptidase (γ-GT) and/or γ-glutamylcyclotransferase (γ-GCT), two enzymes overexpressed in the injured kidney, and evaluated them in in vivo murine models of acute tubular and glomerular damage. Exposure of the animals to disease-inducing drugs together with the functionalized GSIs improved proteinuria and, to some extent, kidney dysfunction. The expression of genes involved in the Notch pathway, acute inflammatory stress responses, and the renin-angiotensin system was enhanced in injured kidneys, which could be downregulated upon administration of functionalized GSIs. Immunohistochemistry staining and Western blots demonstrated enhanced activation of Notch1 as detected by its cleaved active intracellular domain during acute kidney injury, and this was downregulated by concomitant treatment with the functionalized GSIs. Thus targeted γ-secretase-based prodrugs developed as substrates for γ-GT/γ-GCT have the potential to selectively control Notch activation in kidney diseases with subsequent regulation of the inflammatory stress response and the renin-angiotensin pathways.

Activatable Near-Infrared Probe for Fluorescence Imaging of γ-Glutamyl Transpeptidase in Tumor Cells and In Vivo.

γ-Glutamyl transpeptidase (GGT) is a cell-membrane-bound enzyme that is involved in various physiological and pathological processes and is regarded as a potential biomarker for many malignant tumors, precise detection of which is useful for early cancer diagnosis. Herein, a new GGT-activatable near-infrared (NIR) fluorescence imaging probe (GANP) by linking of a GGT-recognitive substrate γ-glutamate (γ-Glu) and a NIR merocyanine fluorophore (mCy-Cl) with a self-immolative linker p-aminobenzyl alcohol (PABA) is reported. GANP was stable under physiological conditions, but could be efficiently activated by GGT to generate ≈100-fold enhanced fluorescence, enabling high sensitivity (detection limit of ≈3.6 mU L-1 ) and specificity for the real-time imaging of GGT activity as well as rapid evaluation of the inhibition efficacy of GGT inhibitors in living tumor cells. Notably, the deep tissue penetration ability of NIR fluorescence could further allow GANP to image GGT in frozen tumor tissue slices with large penetration depth (>100 µm) and in xenograft tumors in living mice. This GGT activatable NIR fluorescence imaging probe could facilitate the study and diagnosis of other GGT-correlated diseases in vivo.

Synthesis and evaluation of the inhibitory activity of the four stereoisomers of the potent and selective human γ-glutamyl transpeptidase inhibitor GGsTop.

2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (RP/SP). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (kon=174M-1s-1) was ca. 8-fold more potent than the d-isomer (kon=21.5M-1s-1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be SP. The SP-isomers inhibited human GGT (kon=21.5-174M-1s-1), while the RP-isomers were inactive even at concentrations of 0.1mM.

An Activatable Photosensitizer Targeted to γ-Glutamyltranspeptidase.

We adopted a spirocyclization-based strategy to design γ-glutamyl hydroxymethyl selenorhodamine green (gGlu-HMSeR) as a photo-inactive compound that would be specifically cleaved by the tumor-associated enzyme γ-glutamyltranspeptidase (GGT) to generate the potent photosensitizer HMSeR. gGlu-HMSeR has a spirocyclic structure and is colorless and does not show marked phototoxicity toward low-GGT-expressing cells or normal tissues upon irradiation with visible light. In contrast, HMSeR predominantly takes an open structure, is colored, and generates reactive oxygen species upon irradiation. The γ-glutamyl group thus serves as a tumor-targeting moiety for photodynamic therapy (PDT), switching on tumor-cell-specific phototoxicity. To validate this system, we employed chick chorioallantoic membrane (CAM), a widely used model for preliminary evaluation of drug toxicity. Photoirradiation after gGlu-HMSeR treatment resulted in selective ablation of implanted tumor spheroids without damage to healthy tissue.

Pulmonary epithelial cancer cells and their exosomes metabolize myeloid cell-derived leukotriene C4 to leukotriene D4.

Leukotrienes (LTs) play major roles in lung immune responses, and LTD4 is the most potent agonist for cysteinyl LT1, leading to bronchoconstriction and tissue remodeling. Here, we studied LT crosstalk between myeloid cells and pulmonary epithelial cells. Monocytic cells (Mono Mac 6 cell line, primary dendritic cells) and eosinophils produced primarily LTC4 In coincubations of these myeloid cells and epithelial cells, LTD4 became a prominent product. LTC4 released from the myeloid cells was further transformed by the epithelial cells in a transcellular manner. Formation of LTD4 was rapid when catalyzed by γ-glutamyl transpeptidase (GGT)1 in the A549 epithelial lung cancer cell line, but considerably slower when catalyzed by GGT5 in primary bronchial epithelial cells. When A549 cells were cultured in the presence of IL-1ß, GGT1 expression increased about 2-fold. Also exosomes from A549 cells contained GGT1 and augmented LTD4 formation. Serine-borate complex (SBC), an inhibitor of GGT, inhibited conversion of LTC4 to LTD4 Unexpectedly, SBC also upregulated translocation of 5-lipoxygenase (LO) to the nucleus in Mono Mac 6 cells, and 5-LO activity. Our results demonstrate an active role for epithelial cells in biosynthesis of LTD4, which may be of particular relevance in the lung.

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.

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.

GGsTop, a novel and specific γ-glutamyl transpeptidase inhibitor, protects hepatic ischemia-reperfusion injury in rats.

Ischemia-reperfusion (IR) injury is a major clinical problem and is associated with numerous adverse effects. GGsTop [2-amino-4{[3-(carboxymethyl)phenyl](methyl)phosphono}butanoic acid] is a highly specific and irreversible γ-glutamyl transpeptidase (γ-GT) inhibitor. We studied the protective effects of GGsTop on IR-induced hepatic injury in rats. Ischemia was induced by clamping the portal vein and hepatic artery of left lateral and median lobes of the liver. Before clamping, saline (IR group) or saline containing 1 mg/kg body wt of GGsTop (IR-GGsTop group) was injected into the liver through the inferior vena cava. At 90 min of ischemia, blood flow was restored. Blood was collected before induction of ischemia and prior to restoration of blood flow and at 12, 24, and 48 h after reperfusion. All the animals were euthanized at 48 h after reperfusion and the livers were harvested. Serum levels of alanine transaminase, aspartate transaminase, and γ-GT were significantly lower after reperfusion in the IR-GGsTop group compared with the IR group. Massive hepatic necrosis was present in the IR group, while only few necroses were present in the IR-GGsTop group. Treatment with GGsTop increased hepatic GSH content, which was significantly reduced in the IR group. Furthermore, GGsTop prevented increase of hepatic γ-GT, malondialdehyde, 4-hydroxynonenal, and TNF-α while all these molecules significantly increased in the IR group. In conclusion, treatment with GGsTop increased glutathione levels and prevented formation of free radicals in the hepatic tissue that led to decreased IR-induced liver injury. GGsTop could be used as a pharmacological agent to prevent IR-induced liver injury and the related adverse events.

Platelet protective efficacy of 3,4,5 trisubstituted isoxazole analogue by inhibiting ROS-mediated apoptosis and platelet aggregation.

Thrombocytopenia is a major hematological concern in oxidative stress-associated pathologies and chronic clinical disorders, where premature platelet destruction severely affects the normal functioning of thrombosis and hemostasis. In addition, frequent exposure of platelets to chemical entities and therapeutic drugs immensely contributes in the development of thrombocytopenia leading to huge platelet loss, which might be fatal sometimes. Till date, there are only few platelet protective molecules known to combat thrombocytopenia. Hence, small molecule therapeutics are extremely in need to relieve the burden on limited treatment strategies of thrombocytopenia. In this study, we have synthesized a series of novel 3,4,5 trisubstituted isoxazole derivatives, among which compound 4a [4-methoxy-N'-(5-methyl-3-phenylisoxazole-4-carbonyl) benzenesulfonohydrazide] was found to significantly ameliorate the oxidative stress-induced platelet apoptosis by restoring various apoptotic markers such as ROS content, cytosolic Ca(2+) levels, eIF2-α phosphorylation, mitochondrial membrane depolarization, cytochrome c release, caspase activation, PS externalization, and cytotoxicity markers. Additionally, compound 4a dose dependently inhibits collagen-induced platelet aggregation. Hence, compound 4a can be considered as a prospective molecule in the treatment regime of platelet activation and apoptosis and other clinical conditions of thrombocytopenia. Further studies might ensure the use of compound 4a as a supplementary therapeutic agent to treat, thrombosis and CVD-associated complications. Over all, the study reveals a platelet protective efficacy of novel isoxazole derivative 4a with a potential to combat oxidative stress-induced platelet apoptosis.

Phosphonate-based irreversible inhibitors of human γ-glutamyl transpeptidase (GGT). GGsTop is a non-toxic and highly selective inhibitor with critical electrostatic interaction with an active-site residue Lys562 for enhanced inhibitory activity.

γ-Glutamyl transpeptidase (GGT, EC 2.3.2.2) that catalyzes the hydrolysis and transpeptidation of glutathione and its S-conjugates is involved in a number of physiological and pathological processes through glutathione metabolism and is an attractive pharmaceutical target. We report here the evaluation of a phosphonate-based irreversible inhibitor, 2-amino-4-{[3-(carboxymethyl)phenoxy](methoyl)phosphoryl}butanoic acid (GGsTop) and its analogues as a mechanism-based inhibitor of human GGT. GGsTop is a stable compound, but inactivated the human enzyme significantly faster than the other phosphonates, and importantly did not inhibit a glutamine amidotransferase. The structure-activity relationships, X-ray crystallography with Escherichia coli GGT, sequence alignment and site-directed mutagenesis of human GGT revealed a critical electrostatic interaction between the terminal carboxylate of GGsTop and the active-site residue Lys562 of human GGT for potent inhibition. GGsTop showed no cytotoxicity toward human fibroblasts and hepatic stellate cells up to 1mM. GGsTop serves as a non-toxic, selective and highly potent irreversible GGT inhibitor that could be used for various in vivo as well as in vitro biochemical studies.

Direct Monitoring of γ-Glutamyl Transpeptidase Activity In Vivo Using a Hyperpolarized (13) C-Labeled Molecular Probe.

The γ-glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ-Glu-[1-(13) C]Gly, for monitoring GGT activity in vivo by hyperpolarized (HP) (13) C magnetic resonance (MR). The properties of γ-Glu-[1-(13) C]Gly are suitable for in vivo HP (13) C metabolic analysis since the chemical shift between γ-Glu-[1-(13) C]Gly and its metabolic product, [1-(13) C]Gly, is large (4.3 ppm) and the T1 of both compounds is relatively long (30 s and 45 s, respectively, in H2 O at 9.4 T). We also demonstrate that γ-Glu-[1-(13) C]Gly is highly sensitive to in vivo modulation of GGT activity induced by the inhibitor acivicin.

Effect of arsenite on nitrosative stress in human breast cancer cells and its modulation by flavonoids.

Arsenic (As) is used in the treatment of leukemia and breast cancer due to its oxidative cytotoxic action. However, it is also toxic to normal cells. One proposed anticancer mechanism induced by As might be nitrosative stress (NS). It is believed that antioxidant flavonoids in combination with As might reduce its toxic action on normal cells without interfering with its antitumor action. In the present study, we evaluated the antineoplastic potential of As on breast human cancer lines MCF-7 and ZR-75-1 treated with redox-modulating flavonoids, such as quercetin (Q) and silymarin (S). Even though both cell lines differed about their oxidative responsiveness, their viability was decreased by NS induction through γ-glutamyltranspeptidase inhibition. Arsenic triggered NS in both MCF-7 and ZR-75-1 cultures, with the formers more sensitive without recovering their pre-treatment capacity. ZR-75-1 cells maintained their antioxidant status, whereas MCF-7 ones treated with S, As, and As + Q did not. Silymarin did not interfere with the described As bioactivity. NS was an anticancer mechanism exerted by As depending on the redox cellular response that could be differentially modified by dietary antioxidants. Hence, it is worthwhile to consider the use of dietary antioxidants as adjuvant in cancer chemotherapy, especially when using As.

A reaction path study of the catalysis and inhibition of the Bacillus anthracis CapD γ-glutamyl transpeptidase.

The CapD enzyme of Bacillus anthracis is a γ-glutamyl transpeptidase from the N-terminal nucleophile hydrolase superfamily that covalently anchors the poly-γ-D-glutamic acid (pDGA) capsule to the peptidoglycan. The capsule hinders phagocytosis of B. anthracis by host cells and is essential for virulence. The role CapD plays in capsule anchoring and remodeling makes the enzyme a promising target for anthrax medical countermeasures. Although the structure of CapD is known, and a covalent inhibitor, capsidin, has been identified, the mechanisms of CapD catalysis and inhibition are poorly understood. Here, we used a computational approach to map out the reaction steps involved in CapD catalysis and inhibition. We found that the rate-limiting step of either CapD catalysis or inhibition was a concerted asynchronous formation of the tetrahedral intermediate with a barrier of 22-23 kcal/mol. However, the mechanisms of these reactions differed for the two amides. The formation of the tetrahedral intermediate with pDGA was substrate-assisted with two proton transfers. In contrast, capsidin formed the tetrahedral intermediate in a conventional way with one proton transfer. Interestingly, capsidin coupled a conformational change in the catalytic residue of the tetrahedral intermediate to stretching of the scissile amide bond. Furthermore, capsidin took advantage of iminol-amide tautomerism of its diacetamide moiety to convert the tetrahedral intermediate to the acetylated CapD. As evidence of the promiscuous nature of CapD, the enzyme cleaved the amide bond of capsidin by attacking it on the opposite side compared to pDGA.

Structure of Bacillus subtilis γ-glutamyltranspeptidase in complex with acivicin: diversity of the binding mode of a classical and electrophilic active-site-directed glutamate analogue.

γ-Glutamyltranspeptidase (GGT) is an enzyme that plays a central role in glutathione metabolism, and acivicin is a classical inhibitor of GGT. Here, the structure of acivicin bound to Bacillus subtilis GGT determined by X-ray crystallography to 1.8 Šresolution is presented, in which it binds to the active site in a similar manner to that in Helicobacter pylori GGT, but in a different binding mode to that in Escherichia coli GGT. In B. subtilis GGT, acivicin is bound covalently through its C3 atom with sp2 hybridization to Thr403 Oγ, the catalytic nucleophile of the enzyme. The results show that acivicin-binding sites are common, but the binding manners and orientations of its five-membered dihydroisoxazole ring are diverse in the binding pockets of GGTs.

Glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of γ-glutamyl transpeptidase for probing cysteinyl-glycine binding site.

γ-Glutamyl transpeptidase (GGT) catalyzing the cleavage of γ-glutamyl bond of glutathione and its S-conjugates is involved in a number of physiological and pathological processes through glutathione homeostasis. Defining its Cys-Gly binding site is extremely important not only in defining the physiological function of GGT, but also in designing specific and effective inhibitors for pharmaceutical purposes. Here we report the synthesis and evaluation of a series of glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of human and Escherichia coli GGTs to probe the structural and stereochemical preferences in the Cys-Gly binding site. Both enzymes were inhibited strongly and irreversibly by the peptidyl phosphorus esters with a good leaving group (phenoxide). Human GGT was highly selective for l-aliphatic amino acid such as l-2-aminobutyrate (l-Cys mimic) at the Cys binding site, whereas E. coli GGT significantly preferred l-Phe mimic at this site. The C-terminal Gly and a l-amino acid analogue at the Cys binding site were necessary for inhibition, suggesting that human GGT was highly selective for glutathione (γ-Glu-l-Cys-Gly), whereas E. coli GGT are not selective for glutathione, but still retained the dipeptide (l-AA-Gly) binding site. The diastereoisomers with respect to the chiral phosphorus were separated. Both GGTs were inactivated by only one of the stereoisomers with the same stereochemistry at phosphorus. The strict recognition of phosphorus stereochemistry gave insights into the stereochemical course of the catalyzed reaction. Ion-spray mass analysis of the inhibited E. coli GGT confirmed the formation of a 1:1 covalent adduct with the catalytic subunit (small subunit) with concomitant loss of phenoxide, leaving the peptidyl moiety that presumably occupies the Cys-Gly binding site. The peptidyl phosphonate inhibitors are highly useful as a ligand for X-ray structural analysis of GGT for defining hitherto unidentified Cys-Gly binding site to design specific inhibitors.