Selective inhibition of gamma-glutamyl-cycle enzymes by substrate analogs.

Abstract

Substrate analogs have been obtained that selectively inhibit the reactions of the -glutamyl cycle or that are susceptible to only limited metabolism by the cycle. Thus, glutathione synthesis may be inhibited and analogs of glutathione may be synthesized that do not participate in transpeptidation. Specific inhibitors of "y-glutamylcyclotransferase and 5-oxoprolinase have been obtained. The findings offer new approaches to the in vivo study of the cycle and also to the design of more specifically directed analogs of inhibitors such as methionine sulfoximine and 6-diazo-5-oxonorleucine.The enzymic reactions of the y-glutamyl cycle [which accounts for the synthesis and degradation of glutathione ( Fig. 1) (1)] include synthesis of the "y-glutamyl peptide bond, transfer of the y-glutamyl group to an acceptor, cyclization of the -y-glutamyl group to form 5-oxoproline, and energy-dependent decyclization of 5-oxoproline to glutamate. In an approach to further understanding of the function of the cycle, we have sought specific inhibitors of its individual reactions and also substrate analogs that would function in some but not all of the reactions. Such goals might be difficult to achieve because all of these reactions involve the y-carboxyl group of glutamate, and a given substrate analog might therefore be expected to interact with more than one enzyme. However, the several enzymic reaction pathways are clearly different and are presumably associated with differences in the enzyme-bound conformations of the glutamate carbon chain at the active sites of the several enzymes.In the present work, we have attempted to exploit such expected differences among the enzymes by manipulation of substrate structure.* We have found that suitable modification of the glutamyl moiety of the substrates yields the desired results: effective and specific inhibitors or nonmetabolizable analogs at each of the steps of the 'y-glutamyl cycle. The present findings thus offer several potentially new approaches to the study of the cycle in vvo and also provide information relevant to further investigations on the mechanisms of action of these metabolically related enzymes. The data reported here may be applied to the modification of potent (but relatively nonspecific) inhibitors such as methionine sulfoximine, azaserine, and 6-diazo-5-oxonorleucine so as to achieve one-enzyme specificity. The findings also make possible improved assay procedures for y-glutamyl-cysteine synthetase, glutathione synthetase, and y-glutamylcyclotransferase in which the substrates and products are not degraded by other enzymes present in unpurified tissue extracts. D-y-Glutamyl-L-a-aminobutyric acid, 'y-(y-methyl)glutamyl-L-a-aminobutyric acid, and fl-aminoglutaryl-L-a-aminobutyric acid were prepared via, the corresponding Nphthaloyl glutamic anhydride analogs as described (7). L-'y-glutamyl-La-aminobutyric acid, and f3-aminoglutaryl-L-a-aminobutyric acid were prepared enzymatically with -y-glutamyl-cysteine synthetase by using phosphoenolpyruvate and pyruv...

The mercapturic acid pathway

“…Substrate selectivity studies with rat kidney GGT1 show that recognition of the a-amino group and the pK a of the free proton of the c-peptide bond are critical determinants of donor substrate selectivity (Cook et al 1987). Both L-and D-c-Glu compounds are substrates (Tate and Meister 1985); alkyl groups in the side-chain of the c-Glu moiety are either poorly tolerated or not tolerated (Griffith and Meister 1977), and replacement of the 4-methylene of the c-Glu moiety with heteroatoms yields inactive donor substrates (Lherbet et al 2003). Moreover, c-Glu-4-nitrophenyl ester, the ester analog of the widely used assay substrate c-Glu-4-nitroanilide (196, Figure 19), is not a substrate (Castonguay et al 2002).…”

A glutamine synthetase inhibitor increases survival and decreases cytokine response in a mouse model of acute liver failure

“…γ‐glutamyl cysteine synthetase was assayed in the same extracts as described by Griffith and Meister (21).…”

Design and evaluation of novel analogs of 2-amino-4-boronobutanoic acid (ABBA) as inhibitors of human gamma-glutamyl transpeptidase

“…2 ). 22 – 31 Several crystals structures have been solved of prokaryotic GGT1 with these inhibitors bound in the active site. 32 – 34 We have published the only structures of hGGT1 including structures with glutamate analogs that inhibit GGT1 bound in the active site., 19 , 35 , 36 Both the prokaryotic and human GGT1 structures show that the glutamate moiety of the inhibitors occupies the active site with the same orientation as the gamma-glutamyl group of glutathione.…”