Cysteine synthase thiols

This enzyme [EC 4.2.99.8], also known as cysteine synthase and O-acetylserine sulfhydrylase, catalyzes the pyr-idoxal-phosphate-dependent reaction of H2S with O -acetylserine to produce cysteine and acetate. Some alkyl thiols, cyanide, pyrazole, and some other heterocyclic compounds can also act as acceptors. 

Fig. 3. Regulation of the bound pathway for the assimilation of sulfate into cysteine and associated processes. Carrier refers to an endogenous thiol of uncertain identity in higher plants. Enzymes associated with the sulfate assimilation pathway and the synthesis of O-acetylseiine are (1) high-ailinity sulfate uptake mechanism, (2) ATP-sulfurylase, (3) adenosine S -phosphosulfate (APS) sulfotransferase, (4) organic thiosulfate reductase, (5) cysteine synthase, and (6) serine transacetylase. Cysteine sulfhydrase (7), an enzyme of cysteine catabolism, and nitrate reductase (8), the first enzyme of the nitrate assimilation pathway, are also shown. Inhibitory control of the pathways is shown by discontinuous lines (----) and enhancement by continuous lines (------).

Figure 21-2. Fatty acid synthase multienzyme complex. The complex is a dimer of two identical polypeptide monomers, 1 and 2, each consisting of seven enzyme activities and the acyl carrier protein (ACP). (Cys— SH, cysteine thiol.) The— SH of the 4 -phosphopantetheine of one monomer is in close proximity to the— SH of the cysteine residue of the ketoacyl synthase of the other monomer, suggesting a "head-to-tail" arrangement of the two monomers. Though each monomer contains all the partial activities of the reaction sequence, the actual functional unit consists of one-half of one monomer interacting with the complementary half of the other. Thus, two acyl chains are produced simultaneously. The sequence of the enzymes in each monomer is based on Wakil.

The fatty acid synthase protein is known to contain an acyl carrier protein (ACP) binding site, and also an active-site cysteine residue in the P-ketoacyl synthase domain. Acetyl and malonyl gronps are successively transferred from coenzyme A esters and attached to the thiol groups of Cys and ACP. 

This pyridoxal-phosphate-dependent enzyme [EC 4.2.99.9], also known as cystathionine y-synthase, catalyzes the reaction of O-succinyl-L-homoserine with L-cysteine to produce cystathionine and succinate. The enzyme can also use hydrogen sulfide and methanethiol as substrates, producing homocysteine and methionine, respectively. In the absence of a thiol, the enzyme can also catalyze a /3,y-elimination reaction to form 2-oxobu-tanoate, succinate, and ammonia. 

Nitric oxide generation from L-arginine and nitric oxide donors and the formation of cGMP. L-NMMA inhibits nitric oxide synthase. Some of the nitric oxide donors such as furoxans and organic nitrates and nitrites require a thiol cofactor such as cysteine or glutathione to form nitric oxide. 

Following loading of acetyl and malonyl groups onto the P subunit of the enzyme, additional intramolecular transfers must occur to prepare the substrates for the decarboxylative condensation reaction which is catalyzed by the -ketoacyl synthase domain of the a subunit. The end result of these transfers is the thio-esterification of malonate by the phosphopantetheine thiol and of acetate by Cys-1305(a) of the -keto synthase active site. This cysteine has been shown to have a dramatically lowered pK (<5), which would encourage its reactivity [65]. 

Elegant experiments, which capitalized on the ability of iodoacetamide to specifically alkylate the active site cysteine of the -ketoacyl synthase, were performed, which definitively proved the capability of the yeast FAS in decar-boxylating malonyl CoA [75]. Following alkylation, FAS activity is abolished however, the enzyme still transacylates malonyl CoA to the phosphopantetheine thiol, where it is decarboxylated before being transferred back to CoA by the transacylase prior to its release as acetyl CoA. 

Substrates are processed directly through cysteine thiols, and cerulenin and iodoacetamide are able to inhibit the activity of plant PKSs by modifying sulf-hydryl groups of these residues [ 134]. Although chalcone and stilbene synthases do not share a high level of overall sequence similarity with FASs and PKSs, the cysteine that is modified by cerulenin is conserved, suggesting that the active sites may be structurally related [51]. 

GSH synthase, a tripeptide (glutamylcysteinylglycine), is not only a water-soluble antioxidant, but is also part of a redox buffer (Smith et al., 1996). It is found in all cells and is used for a multiplicity of cellular functions, such as protein and prostaglandin synthesis, detoxification, etc. Cytosolic concentrations of GSH range from 1 to 11 mm (Smith et al., 1996) and are 100-1000 times greater than the extracellular levels. Many proteins contain sulfhydryl groups because of their cysteine content. The content of thiols in proteins is greater than that of the pool of GSH (Torchinsky, 1981). 

Several PLP-dependent enzymes catalyze elimination and replacement reactions at the y-carbon of substrates, an unusual process which provides novel routes for mechanism-based inactivation. An example of this class of enzymes is cystathionine y-synthase [0-succinylhomoserine (thiol)-lyase], which converts (7-succinyl-L-homoserine and L-cysteine to cystathionine and succinate as part of the bacterial methionine biosynthetic pathway (Walsh, 1979, p. 823). Formation of a PLP-stabilized o-carbanion intermediate activates the )8-hydrogen for abstraction, yielding j8-carbanion equivalents and allowing elimination of the y-substituent. The resulting j8,y-unsaturated intermediate serves as an electrophilic acceptor for the replacement nucleophile. Suitable manipulation of the j8-carbanion intermediate allows strategies for the design of inactivators which do not affect enzymes which abstract only the a-hydrogen. 

Such a condensation is mediated by the enzyme 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS). The mechanism of this catalysis is outlined in Figure 1.21. An initial frani-thioesterase step transfers the acetyl group of the first acetyl-CoA to an enzymatic cysteine. In the Claisen condensation phase of the reaction, the a-carbon of a second acetyl-CoA is deprotonated, forming an enolate. The enolate carbon attacks the electrophilic thioester carbon, forming a tetrahedral intermediate which quickly collapses to expel the cysteine thiol [22]. 

There are some known inhibitors of fatty acid synthesis. Cerulenin inhibits KAS I completely and irreversibly at 20 j.M [5] and inhibits KAS II but at higher concentrations. Cerulenin also inhibits the condensing enzyme function of multifunctional protein type I fatty acid synthases [6]. For all these enzymes the mechanism of action Is similar, involving the covalent binding of the inhibitor to the cysteine of the active site of the protein [7]. Arsenite, an inhibitor of enzymes containing vicinal thiol groups, inhibits KAS II but no other condensing enzyme. KAS III is insensitive to inhibition by either cerulenin or arsenite but its activity is inhibited by thiolactomycin (TLM). This inhibition has been seen in bacteria [8] and plants [9] but is rather variable as reported in the literature [10-14]. 

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