Thiol esterases

A particular interest for clinical applications was a possibility for detection of dopamine by its oxidation on nickel [19], cobalt [65], and osmium [66] hexacyanofer-ates. Except for oxidation of dopamine, cobalt and osmium hexacyanoferrates were active in oxidation of epinephrine and norepinephrine. For clinical analysis it is also important to carry out the detection of morphine on cobalt [67] and ferric [68] hexacyanoferrates, as well as the detection of oxidizable amino acids (cystein, methionine) by manganous [69] and ruthenium [70] hexacyanoferrate-modified electrodes. In general, oxidation of thiols was first shown for Prussian blue [71] and nickel hexacyanoferrate [72], This approach has been used for the detection of thiols in rat striatum microdialysate [73], Alternatively, the detection of thiocholine with Prussian blue was employed for pesticide determination in acetylcholine-esterase test [74],... 

In conclusion, sydnonimines represent a class of NO-donors that, with the exception of N-acylated derivatives that need chemical hydrolysis or enzymatic activation by esterases, release NO spontaneously in the presence of oxidants without requiring further activation with enzymes or thiols. 

There are few drugs that are thioesters, but you may recall that one of the intermediates in the oxidation of aldehydes by aldehyde dehydrogenase is a thioester involving the thiol of the enzyme (Fig. 30 in Chapter 4), which is readily hydrolyzed back to the native form of the enzyme, a thiol, and the carboxylic acid product. Some drugs that are carboxylic acids, such as enaloprilate, are administered as ester prodrugs (enalopril), which are more readily absorbed from the intestine than the carboxylic acid and are then readily hydrolyzed to the active drug by esterases as mentioned in Chapter 1 (Fig. 1 in Chapter 1). 

In enzymes, the most common nucleophilic groups that are functional in catalysis are the serine hydroxyl—which occurs in the serine proteases, cholinesterases, esterases, lipases, and alkaline phosphatases—and the cysteine thiol—which occurs in the thiol proteases (papain, ficin, and bromelain), in glyceraldehyde 3-phosphate dehydrogenase, etc. The imidazole of histidine usually functions as an acid-base catalyst and enhances the nucleophilicity of hydroxyl and thiol groups, but it sometimes acts as a nucleophile with the phos-phoryl group in phosphate transfer (Table 2.5). 

The second pK, 8.5-9.5, derived from the pH-activity curves, is much more difficult to interpret. This pK is naturally absent in the system imidazol + ester (21). It is also subject to much greater variation than pK0. This has been demonstrated for a variety of substrates (Fig. 3), but is especially prominent when thiol esters are being studied (Figs. 4 and 5). In the system eel esterase-acetylthiocholine, no decrease of activity is observed on the alkaline side up to pH 11, and for plasma cholinesterase-acetylthiocholine the decrease is very much delayed, when compared with the oxy ester, acetylcholine (see Fig. 2). Similar observations have been made with other esterases and other thiol esters (44)- They indicate that the second component 02 of the esteratic site, to which pK has to be ascribed, may be less essential for certain substrates than for others. 

A second function of NAD+ in the reaction is unrelated to its redox role since it is required as a cofactor for most of the nonoxidative reactions involving the reactive thiol group, with the exception of the esterase activity (13, 54, 184-188). 

Although NAD inhibits the esterase reaction, adenine nucleotides stimulate it, which suggests that NAD is blocking access of the aromatic ester to the reactive thiol in a manner similar to its effect on iodoaceta-mide alkylation (70). Acyl shifts involving Cys-149 and Lys-183 also occur readily with p-nitrophenyl acetate at higher pH and in the absence of NAD (70, 73, 74). 

The yeast strain used for fermentation had no impact on the enantiomer distribution of these volatile thiols. 3SHA is generally considered to be formed by esterification of 3SH by yeast during alcoholic fermentation. The esterase or lipase involved probably acetylates 3SH with a certain enantioselectivity. In contrast, the enantiomer distribution of 3SH in wine made from botrytized grapes (Botrytis cinerea) is 25 75 in favor of the S form, which has also been found in botrytized must (Thibon et al. 2007,2008a). 

The inhibition of hLAL by boronic acids and diethyl p-nitrophenyl phosphate (Sando and Rosenbaum, 1985 G. N. Sando and H. L. Brockman, unpublished, cited by Anderson and Sando, 1991) indicates that hLAL is a serine hydrolase. Two lipase/esterase consensus pentapep-tides, G-X-S-X-G, are found, but only one of them appears to be consistent with the packing requirements of the )8-eSer-a nucleophilic motif (see above). Susceptibility of the enzyme to sulfhydryl reagents, and the requirement of thiols for the stability of purified hLAL, prompted Anderson and Sando (1991) to propose that a cysteine residue, or rather a Cys/Ser couple, may be involved in an internal transacylation reaction. It must be pointed out, however, that hLAL has all three cysteines of the gastric enzyme (as well as six additional ones), and so the inhibitory Cys is also there. The same argument proposed herein with respect to hGL, i.e., that a free cysteine is topologically close to the active site, also holds for hLAL. 

Thioesters are hydrolysed by lipases and esterases (Kurooka et al., 1976) the rate of hydrolysis increases as the length of the carbon chain increases and decreases as the oxygenation of the carbon chain in the thiol moiety increases (Greenzaid Jenks, 1971). After hydrolysis, the resulting alcohol and carboxylic acid would participate in the metabolic pathways described above for sulfides containing oxygenated functional groups. 

Kurooka, S., Hashimoto, M., Tomita, M., Maki, A. Yoshimura, Y. (1976) Relationship between the structure of S-acyl thiol compounds and their rates of hydrolysis by pancreatic lipase and hepatic carboxylic esterase. J. Biochem. 79, 533-541. 

The mechanism of amide- and ester-hydrolyzing enzymes is very similar to that observed in the chemical hydrolysis by a base. A nucleophilic group from the active site of the enzyme attacks the carbonyl group of the substrate ester or amide. This nucleophilic chemical operator can be either the hydroxy group of a serine (e.g., pig fiver esterase, subtifisin, and the majority of microbial lipases), a carboxyl group of an aspartic acid (e.g., pepsin) [3], or the thiol functionality of cysteine (e.g., papain) [4-6]. 

Stereospecific Michael addition reactions also may be catalyzed by hydrolytic enzymes (Scheme 2.205). When ot-trifluoromethyl propenoic acid was subjected to the action of various proteases, lipases and esterases in the presence of a nucleophile (NuH), such as water, amines, and thiols, chiral propanoic acids were obtained in moderate optical purity [1513]. The reaction mechanism probably involves the formation of an acyl enzyme intermediate (Sect. 2.1.1, Scheme 2.1). Being an activated derivative, the latter is more electrophilic than the free carboxylate and undergoes an asymmetric Michael addition by the nucleophile, directed by the chiral environment of the enzyme. In contrast to these observations made with crude hydrolase preparations, the rational design of a Michaelase from a lipase-scaffold gave disappointingly low stereoselectivities [1514-1517]. 

Esterases a large group of hydrolases acting on ester bonds R CXTOR -f HjO - R COOH-r R OH. The acid may be a carboxylic acid, phosphoric or sulfuric acid. The ester may be an alcoholic or a thiol ester. 

The thiol enzyme for which the most detailed mechanistic formulations have been proposed is papain . In this enzyme a cysteine thiol group appears to function in the same manner as the serine hydroxyl of other proteases and esterases. In the hydrolysis of proteins by this plant protease there is an intermediate formation of an acyl thiol, which is subsequently cleaved by water. 

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