Thiol substrate specificity

Various sulfur-containing compounds, including thioamides, thioureas, thiols, thioethers and disulfides, are oxidized by this enzyme system. However, unlike cytochromes P-450, it cannot catalyze hydroxylation reactions at carbon atoms. It is clear that this enzyme system has an important role in the metabolism of xenobiotics, and examples will appear in the following pages. Just as with the cytochromes P-450 system, there appear to be a number of isoenzymes, which exist in different tissues, which have overlapping substrate specificities. 

The discovery of non-specific disulfide reductases which are labile in aerobic cellular extracts suggests that kinetic constraints of thiol/disulfide exchange in vivo are very complex. One of such proteins is thioredoxin which behaves as a non-specific protein-disulfide reductase. Thioredoxin also works as a cofactor of sulfoxide reductases. The dithiol active site of thioredoxin sits on a protrusion of the protein surface [274], Thioredoxin is an ubiquitous protein whose molecular weight is about 12 KDa [274,275], It has been found in cytosolic and mitochondrial [276] compartments of animal cells, and it is partly bound to membranes. High contents in thioredoxin have been found in neurons, secretory and epithelial cells. Redox recycling of thioredoxin is insured by thioredoxin reductase, which has been identified in a variety of mammalian cells as a symmetrical dimer with a molecular weight of 116KDa[274]. Thioredoxin reductase is NADPH-specific, but it exhibits a very wide disulfide substrate specificity. 

A number of methyltransferases are able tc methylate small molecules (46,47). Thus, re. actions of methylation fulfill only two of the three criteria defined above, because the methyl group is small compared with the substrate. The main enzyme responsible for O-methylation is catechol 0-methyltransferas (EC 2.1.1.6 COMT), which is mainly cytosolic but also exists in membrane-bound form. Several enzymes catalyze reactions of xenobiotic N-methylation with different substrate specificities, e.g., nicotinamide iV-methyltrans-ferase (EC 2.1.1.1), histamine methyltrans-ferase (EC 2.1.1.8), phenylethanolamine N-methy 1 transferase (noradrenal ine A-meth-yltransferase EC 2.1.1.28), and nonspecific amine N-methyltransferase (arylamine N-methyltransferase, tryptamine JV-methyl-transferase EC 2.1.1.49) of which some isozymes have been characterized. Reactions of xenobiotic S-methylation are mediated by the membrane-bound thiol methyltransferase (EC 2.1.1.9) and the cytosolic thiopurine methyltransferase (EC 2.1.1.67) (3). 

The thiol-template mechanism is utilized in other enzymes involved in production of peptide-based antibiotics. Actinomycin synthetase II (ACMSII) and b-L-(a-aminoadipolyl)-L-cysteinyl-D-valine (ACV) synthetase catalyze the stereoinversion of valine residues vithin peptide-based antibiotics, and employ ATP and the PAN cofactor in a mechanism similar to that depicted in Fig. 7.14 [58, 59]. ACMSII catalyzes the stereoinversion of a valine within the tripeptide 4-MHA-L-Thr-D-Val (MHA, 4-methyl-3-hydroxyanthranilic acid), which is a precursor for the antibiotic actinomycin D. ACV synthetase catalyzes the stereoinversion of the valine within ACV, which is a precursor for penicillin and cephalosporin [60-63]. ACV synthetase has been shown to have much broader substrate specificity, also accepting non-natural substrates [64, 65]. 

CGS catalyzes in vitro a variety of other nonphysiological reactions such as /3-replacement, a-, or /3-proton-exchange reactions and CBL-like /3-elimination reactions, with very low efficiency. In vitro, CGS is able to accept sulfide as the nucleophilic substrate in place of L-Cys, thus maintaining the original sulfhydrylase activity. A microbial CGS exhibits a broad substrate specificity because several thiol compounds such as sodium sulfide, homocysteine, and various alkyl and aryl mercaptans can replace cysteine. ... 

Ribosomal protein S6 from Escherichia coli undergoes a unique post-translation modification where up to six glutamic acid residues are ligated to the C-terminus. [82-84] RimK, also a member of carboxylate-amine/thiol Ugase superfamily, mediated this post-translahonal modification. [84] In vitro analysis of RimK s)rnthesis resulted in 46-mer (maximum length) of a-poly (L-Glu) at pH 9.0, 30 °C. The maximum chain length was pH dependent. Furthermore, RimK demonstrated strict substrate specificity for Glu. [72]... 

Thermolysin (EC 3.4.24.4) a heat-stable, zinc- and calcium-containing neutral protease, M, 37,500, from Bacillus thermoproteolyticus, with a substrate specificity similar to that of Subtilisin (see). After one hour at 80 °C, T. still has 50% original activity. This high heat stability of T. is attributed to the large number of hydrophobic regions and the presence of four bound calcium ions, which serve in place of disulfide bridges (T. contains no disulfide bridges) to maintain the compact shape of the molecule. T. is neither a thiol nor a serine enzyme. 

Another way thiols can participate in enzyme reactions is by binding substrates or coenzymes at the active site. A clear differentiation between involvement in catalytic and binding functions is seldom possible, but a binding role is presumed when protection of the critical thiol is afforded by the presence of substrate and no specific catalytic role is suspected. There are only a few proven examples of thiol substrate binding other than those already discussed in which a precise catalytic role is also proposed. 

The results presented thus far show that substrate specificity at the second elongation step exceeds that of acyl loading also highlight the potential problem of KS enzymatic stalling. In order to restore enzymatic activity either the acyl chain must hydrolyse off the active site Cys, or the upstream ACP must de-acylate the KS in a reversal of the initial acylation step. Once on the upstream ACP, the acyl chain may hydrolyse off the PPant arm, possibly catalysed by an acyl hydrolase (AH) domain (Scheme 6.2) [13]. In theory, both mechanisms are plausible, but there is a lack of any clear evidence in the literature for reversibility of the KS acylation step, other than the not unsurprising observation that a large excess of SNAC thiol was able to remove an acyl group from the KS active site by transthioesterification [6]. 

The detrimental effect of bleach should not be underestimated. Some enzymes like papain exhibit several valuable properties for laundering good thermal stability, low substrate specificity. In addition to its detrimental requirement of a low pH for maximum activity, papain contains cysteine residues, which must remain in the thiol form. In the presence of sodium perborate, they are oxidized and the enzyme activity is lost [15]. 

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