P-Lyase

Dekant W, Vamvakas S, Berthold K, et al. 1986c. Bacterial P-lyase mediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcinogenic alkenes trichloroethylene, tetrachloroethylene, and hexachlorobutadiene. Chem Biol Interact 60 31-45. 

C-P lyase has a broad snbstrate specificity and, for example, dimethyl phosphonate is degraded to methane, methylphenyl phosphonate to benzene, and the degradation of the widely nsed herbicide glyphosate may follow alternative pathways both of which involve C-P fission. 

Chen C-M, Q-Z Zhuang, Z Zhu, BL Wanner, CT Walsh (1990) Molecuar biology of carbon-phosphorus bond cleavage Cloning and sequencing of the phn (psiD) genes involved in alkylphosphonate uptake and C-P lyase activity in Escherichia coli. J Biol Chem 265 4461-4471. 

The present chapter reviews applications in biocatalysis of the ONIOM method. The focus is on studies performed in our research group, in most cases using the two-layer ONIOM(QM MM) approach as implemented in Gaussian [23], The studied systems include methane monooxygenase (MMO), ribonucleotide reductase (RNR) [24, 25], isopenicillin N synthase (IPNS) [26], mammalian Glutathione peroxidase (GPx) [27,28], Bi2-dependent methylmalonyl-CoA mutase [29] and PLP-dependent P-lyase [30], These systems will be described in more detail in the following sections. ONIOM applications to enzymatic systems performed by other research groups will be only briefly described. 

Cysteine-S-conjugates have also been proposed as kidney-selective pro-drugs. Renal metabolism of S-6-(purinyl)-L-cysteine resulted in the formation of 6-mercaptopurine by the action of P-lyase [51]. However, besides formation of the intended parent compound, other S-conjugates may be formed by various radical reactions, which may induce renal toxicity. 

Overall, the kidney is highly susceptible to the toxicity of hexachlorobutadiene, in contrast to other organs, due to the activity of p-lyase and other mercapturic acid processing enzymes (Vamvakas et al. 1988b). The greater sensitivity of females may be due to differences in renal enzymes responsible for the tissue levels of the active metabolites (Hook et al. 1983). Based on data in animals, renal toxicity is a major concern in humans who may be chronically exposed to this material from hazardous waste sites or other sources. 

Jones TW, Chen Q, Schaeffer V, et al. 1988. Immunohistochemical localization of glutamine transaminase K, a rat kidney cysteine conjugate p-lyase, and the relationship to the segment specificity of cysteine conjugate nephrotoxicity. Mai Pharm 34 621-627. 

Figure 4.66 Metabolism of a cysteine conjugate by CS lyase (p-lyase). The cysteine conjugate is shown arising from a glutathione conjugate after biliary excretion. The thiol product, which may be toxic (see text) can also be methylated and further oxidized as shown.

Hydrogen sulfide, H2S, is detoxified by methylation first to methanthiol (CH3SH), which is highly toxic, but is then further methylated to dimethylsulfide (CH3-S-CH3). The thiol products of p-lyase may also be methylated by this enzyme. 

Thus, these similar examples illustrate that GSH conjugation may not always lead to a reduction in toxicity. Furthermore, they illustrate that several factors are responsible for the kidney being the target organ active uptake processes, the presence of p-lyase and y-glutamyl transferase, and possibly the importance of mitochondria in the brush border membranes, which are the ultimate target. 

Hexachlorobutadiene is a nephrotoxic industrial chemical, damaging the pars recta of the proximal tubule. Initial conjugation with GSH is necessary, followed by biliary secretion and catabolism resulting in a cysteine conjugate. The conjugate is reabsorbed and transported to the kidney where it can be concentrated and becomes a substrate for the enzyme p-lyase. This metabolizes it into a reactive thiol, which may react with proteins and other critical macro molecules with mitochondria as the ultimate target. The kidney is sensitive because the metabolite is concentrated by active uptake processes (e.g., OAT 1), which reabsorb the metabolite into the tubular cells. 

Other halogenated compounds such as trichloroethylene may be metabolized to similar cysteine conjugates, which are also nephrotoxic but may not all require p-lyase activation. 

Tetrafluoroethylene is metabolized by hepatic glutathione. S -transferase and the resulting cysteine conjugate is further metabolized by renal P-lyase. This pathway results in the formation of a reactive thiol that causes kidney toxicity in rats. 

Dekant, W., Berthold, K., Vamavkas, S., Henschler, D. Anders, M.W. (1988) Thioacylating intermediates as metabolites of S-(l, 2-dichlorovinyl)-L-cysteine and 6 -(l,2,2-trichlorovinyl)-L-cysteine formed by cysteine conjugate P-lyase. Chem. Res. Toxicol., 1, 175-178... 

Cyclohexanedione, reaction with guanidinium groups, 126 Cyclophilin 488 human 488s D-Cycloserine 739s Cyclosporin 488, 488s p Cylinders 65, 66, 686 Cystathionine, 746s formation 746 Cystathionine p lyase 742 Cystathionine p-synthase 744 Cystathionine y-synthase 743, 746 Cystatins 622, 629... 

Cysteine is formed in plants and in bacteria from sulfide and serine after the latter has been acetylated by transfer of an acetyl group from acetyl-CoA (Fig. 24-25, step f). This standard PLP-dependent (3 replacement (Chapter 14) is catalyzed by cysteine synthase (O-acetylserine sulfhydrase).446 447 A similar enzyme is used by some cells to introduce sulfide ion directly into homocysteine, via either O-succinyl homoserine or O-acetyl homoserine (Fig. 24-13). In E. coli cysteine can be converted to methionine, as outlined in Eq. lb-22 and as indicated on the right side of Fig. 24-13 by the green arrows. In animals the converse process, the conversion of methionine to cysteine (gray arrows in Fig. 24-13, also Fig. 24-16), is important. Animals are unable to incorporate sulfide directly into cysteine, and this amino acid must be either provided in the diet or formed from dietary methionine. The latter process is limited, and cysteine is an essential dietary constituent for infants. The formation of cysteine from methionine occurs via the same transsulfuration pathway as in methionine synthesis in autotrophic organisms. However, the latter use cystathionine y-synthase and P-lyase while cysteine synthesis in animals uses cystathionine P-synthase and y-lyase. 

The use of titanium trichloride for the reduction of sulfoxide groups allows a combined approach to the analysis of urine for metabolites derived from hydrolysis and the p-lyase pathway, as shown in Figure 7 (l0 211. Urine is treated with titanium trichloride, divided into two aliquots, and analyzed for TOG and the reduced P-lyase product (10) as described above. 

Cysteine. S -con jugate p-lyase is responsible for converting a number of cysteine S-conjugates into pyruvate, ammonia, and thiols. 

This activity has been demonstrated in mammalian liver and kidney, as well as in intestinal bacteria. Glutathione S-transle rases catalyze the formation of glutathione conjugates, which are processed via the mercapturic biosynthetic pathway, to acety-lated cysteine -conjugates. The unacetylated premercapturic acids are substrates for cysteine 5-conjugate P-lyase, whereas the acetylated cysteine -conjugates, the mercapturic acids, do not function as substrates for the enzymes. 

Cysteine 5-conjugate P-lyase activity has been implicated in the bioactivation of certain halogenated alkenes. This bioactivation results from the generation of reactive thiols. The thiol produced by the P-lyase-dependent metabolism of 5(1,2-dichlorovinyl)-L-cysteine is an unstable electrophile that binds covalently to cellular macromolecules and leads to nephrotoxicity and genotoxicity. Alternatively, certain... 

It is worth noting that background levels of thioglycol and thiodiglycol sulfoxide have been found in the urine of healthy individuals never exposed to sulfur mustard. For this reason their validity as unambiguous markers for sulfur mustard exposure has been questioned. Black and Read (1995) suggested the determination of P-lyase pathway metabolites which in the urine of exposed patients were found at concentrations similar to those of thiodiglycol sulfoxide, but were not found in unexposed individuals. 

Figure 12 Structures of synthetic inhibitors of sphingomyelin synthase, ceramidases, p-glucosylceramide synthase, dihydroceramide desaturase, and SI P lyase.

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