Using a model of hGGT based on the crystal structure of they examined possible binding interactions between GGT and OU749

Using a model of hGGT based on the crystal structure of they examined possible binding interactions between GGT and OU749. or distal benzene ring of the uncompetitive inhibitor, OU749, affected activity. One of the most potent inhibitors was identified among a novel group of analogs with an amine group on the benzosulfonamide ring. New, more potent uncompetitive inhibitors of the physiological GGT reaction were found to be less toxic than the glutamine-analogs that have been tested clinically. Development of non-toxic inhibitors is essential for exploiting GGT as a therapeutic target. is the hydrolysis of the gamma-glutamyl bond. GGT hydrolyzes GSH into glutamate and cysteinylglycine [1, 2]. The gamma-glutamyl substrate (donor substrate) binds to GGT, which initiates a nucleophilic attack on the gamma-glutamyl bond (Fig. 1A). An acyl-bond is formed between the oxygen on the Rabbit Polyclonal to BL-CAM side-chain of Thr-381 of hGGT and the gamma-glutamyl group of the SCH772984 substrate, creating the gamma-glutamyl enzyme intermediate (F-form of the enzyme) [19]. The acyl-bond is hydrolyzed by water to release glutamate and cysteinyl-glycine. The reaction is a modified ping-pong reaction [1]. The addition of high concentrations of dipeptide or amino acid acceptor molecules (second substrate for the ping-pong reaction) results in a transpeptidation reaction (Fig. 1B). The free alpha-amine on the acceptor attacks the acyl-bond, transferring the gamma-glutamyl group to the acceptor, thereby forming a new gamma-glutamyl compound [20]. Open in a separate window Fig. 1 Illustration of the GGT hydrolysis and transpeptidation reactions. The cleavage of GSH is initiated by the nucleophillic attack of the hydroxyl (OH) of the threonine (Thr) on the gamma-glutamyl bond of GSH forming an acyl-bond with the gamma-glutamyl group (-glu) and releasing cysteinylglycine (CysGly) of GSH. The acyl-bond can be either (A) hydrolyzed by water releasing glutamate (Glu) in the hydrolysis reaction or (B) transferred a dipeptide acceptor forming a new gamma-glutamyl group in the transpeptidation reaction. The most commonly used assay for GGT activity monitors the transpeptidation reaction with the synthetic compound L-gamma-glutamyl = 8.6 Hz, 2H), 6.91 (d, = 8.6 Hz, 2H), 7.23 (d, = 8.5 Hz, 2H), 7.35 (d, = 8.6 Hz, 2H), 13.71 (s, 1H). MS(= 8.3 Hz, 2H), 7.39C7.27 (m, 7H). MS(= 8.6 Hz, 2H), 7.32 (dd, = 8.2, 1.8 Hz, 1H), 7.38 (d, = 8.6 Hz, 2H), 7.60 (t, = 8.2 Hz, 1H), 7.64 (d, = 1.7 Hz, 1H). ). MS(= 8.4 Hz, 2H), 7.44C7.30 (m, 6H). MS(= 8.4 Hz, 2H), 7.29C7.10 (m, 4H), 7.42 (ddd, = 20.3, 8.4, 5.7 Hz, 4H). MS(= 8.6 Hz, 2H), 7.18 (t, = 9.2 Hz, 2H), 7.48C7.29 (m, 5H). MS(= 8.2, 3.5 Hz, 4H), 6.94 (d, = 8.4 Hz, 2H), 7.35 (d, = 8.1 Hz, 2H). MS(= 8.4 Hz, 2H), 6.70 (d, = 8.1 Hz, 2H), 7.11 (d, = 8.2 Hz, 2H), 7.35 (d, = 8.3 Hz, 2H). MS(= 8.6 Hz, 2H), 7.14C7.08 (m, 3H), 7.27C7.22 (m, 1H), 7.36 (d, = 8.6 Hz, 2H). MS(= 8.6 Hz, 2H), 7.26 (d, = 8.6 Hz, 2H), 7.49C7.62 (m, 2H), 7.74 (d, = 8.8 Hz, 1H), 7.83C8.05 (m, 3H), 8.17 (s, 1H), 9.00 (s, 1H). Enzyme Isolation hGGT (“type”:”entrez-protein”,”attrs”:”text”:”P19440″,”term_id”:”93140064″,”term_text”:”P19440″P19440), lacking the transmembrane domain, was expressed in and isolated as previously described [17]. The specific activity of the purified GGT was 400 units/mg. One unit of GGT activity was defined as the amount of enzyme that released 1 SCH772984 mol of paranitroaniline/min at 37C at pH 7.4 in the transpeptidation reaction with L-GpNA. L-Glutamate Release Assay (Hydrolysis of GSH) This assay measures the production of glutamate from the hydrolysis of GSH by GGT enzyme and has been described in detail previously [3]. The concentration of the substrate, GSH, was varied from 5 M to 20 M. The concentration of the inhibitors, SCH772984 OU749 and SCH772984 its analogs, were varied from 15.6 M to 250 M. The reaction was initiated with the addition of 10 mU hGGT. The reaction was incubated at 37C and monitored continuously at 490 nm by a Bio-Rad model 680 microplate reader with Microplate Manager 5 software (Bio-Rad, Hercules, CA). All compounds were also evaluated as inhibitors of glutamate dehydrogenase, the enzyme in the second half of the L-glutamate release assay. None of the compounds in this study inhibited glutamate dehydrogenase. L-GpNA Transpeptidation Assay The assay has been described previously [18]. The concentration of the substrate for the transpeptidation reaction, L-GpNA (Sigma, St. Louis, MO) was varied from 0.25 mM to 3 mM in the presence of 40 mM GlyGly (Sigma) as the acceptor. The concentration of L-GpNA was 3 mM for experiments in which the concentration of GlyGly was varied. To initiate the transpeptidation reaction, 4 mU GGT were added. A comparison of the activity of GGT among assays showed that the amount of hGGT that had 10 mU activity in the L-GpNA transpeptidation reaction had 0.868 mU of hGGT activity in the L-Glutamate Release Assay. D-GpNA Hydrolysis Assay The assay was carried out as.