Thiolester hydrolysis

Electrophilic catalysis by Lewis acids is also observed here no ambiguity arises with general acid catalysis, as Lewis acids and proton acids are not the same. An interesting example is the strong catalysis of thiolester hydrolysis by mercuric and silver ions. These soft acids presumably coordinate with the sulfur and, by virtue of the consequent electron withdrawal, make the carbonyl group much more susceptible to attack in the addition mechanism, or, in favorable cases, promote unimolecular SN1 cleavage of the sulfur-carbon bond.122... 

Thiolester hydrolases (EC 3.1.2) play an important role in the biochemistry of lipids. They catalyze the hydrolysis of acyl-coenzyme A thiolesters of various chain lengths to free fatty acids and coenzyme A. The current list of over 20 specific enzymes includes acetyl-CoA hydrolase (EC 3.1.2.1), pal-mi toy 1-Co A hydrolase (EC 3.1.2.2), and an acyl-CoA hydrolase (EC 3.1.2.20) of broad specificity for medium- to long-chain acyl-CoA [128],... 

This enzyme [EC 5.1.1.11], also known as phenylalanine racemase (ATP-hydrolyzing), catalyzes the reaction of ATP with L-phenylalanine to produce o-phenylalanine, AMP, and pyrophosphate. In this unusual racemase reaction, a thiol group of an enzyme-bound pantotheine forms a thiolester from an initial aminoacyl-AMP intermediate then, as is typical of acyl thioesters, the a-proton becomes labile, thereby permitting reversible inversion of configuration to produce an equilibrated mixture of thiolester-bound enantiomers. Hydrolysis of the thiolester yields the product. 

The AG° values for the hydrolysis of any P - O - P bond of ATP, inorganic pyrophosphate, or any acyl CoA thiolester are all about -34 kj / mole, while the corresponding figure for the hydrolysis of a mixed carboxylic phosphate anhydride is about -55 kj / mole. Calculate the value of AG° for the following reaction describing the activation of fatty acids to the fatty acyl adenylate. 

Hydrolase enzymes were initially chosen as target proteins to catalyze and amplify the optimal constituents from dynamic systems. Among the six enzyme classes, hydrolases (EC 3) are one of the most commonly used, both in bulk industrial processes as well as for laboratory scale reactions [1]. These enzymes do not require any cofactors to perform the reactions, and a large variety is commercially available. Hydrolases furthermore catalyze reactions for a broad range of substrates, e.g., hydrolysis of esters, amides, thiolesters, etc., often accompanied with high stereoselectivity. An example of hydrolases is the family of serine hydrolases, which employs... 

After the challenge of the dynamic thiolester system (CDS-1 A) with acetylcholinesterase, the optimal constituents were immediately identified and hydrolyzed to acid and thiol products. The thiol product formed was simultaneously incorporated in the dynamic system to regenerate the optimal thiolester. During the acetylcholinesterase resolution process, two acid products, acetic and propionic acid, respectively, were mainly detected, with the acetyl ester hydrolyzed more rapidly (tl/2 = 210 min) than the propionyl ester (t1/2 = 270 min) as shown in Fig. 2. Only after significant hydrolysis of these two acyl species did the enzyme start to hydrolyze slowly the butyrate thiolester (t1/2 = 1,100 min), a lag phase possibly caused by inhibitory activities of the present thiolesters [8]. All the other acyl groups remained untouched by enzyme, a result which is in accordance with the known specificity of acetylcholinesterase. 

Challenging the dynamic thiolester system CDS-1B with acetylcholinesterase resulted in similar product formations, but a slightly slower enzyme resolution process, than CDS-1A due to the larger size of CDS-1B. To probe the effect of the thiol moiety, dynamic thiolester systems, using only one thiol per system, were furthermore generated and applied to the enzyme resolution process. Thus, the additional CDS-1B, 1C, ID, and IE were prepared with equimolar amounts of five thiolesters 1A-E and one equivalent of either thiol 2, 8,10, or 12 (Table 1). These systems contained ten thiolesters and two thiols. Challenging these systems with acetylcholinesterase resulted in half-lives of formation of the different hydrolysis products, acetic, propionic, and butyric acids, as shown in Table 1. These results indicate that only CDS-1 A (Table 1, Entry 2), generated from five thiolesters 1A-E... 

Esterase Activity. NAD -free GAPDH catalyzes the hydrolysis of aryl esters (e.g., p-nitrophenyl acetate) at rates up to five times that of chymotrypsin (185). This reaction also proceeds via a thiolester intermediate with Cys-149 (99) and is, therefore, inhibited by iodoacetate. 

The hydrolysis of the thiolester occurs by simple substitution at the carbonyl group (Chapter 12). Since a thiol is a good leaving group (pJfa RSH about 7) no strong acid or base is needed. The two bromine atoms will also accelerate nucleophilic attack both because they are electron-withdrawing and because they prevent enolization. 

Applying results from low-molecular-weight thiolesters and esters to the corresponding polymers should give slightly slower acid hydrolysis, equally fast alkaline hydrolysis, and a much faster aminolysis of poly-(thiol esters) compared with polyesters (24, 35, 36). 

There are at least three possible biochemical mechanisms for the activation of carboxyl groups a phosphoric acid anhydride, an adenylate, or a thiolester may be formed. Since radioactivity was not incorporated from either ATP or [2- H adenin]-ATP, into the activated intermediate, a coenz3nne A ester was likely to be present.. Indeed, the activated intermediate was shown to contain coenzyme A and OSB (II) in the ratio 1 1. Thus, hydrolysis of the activated intermediate and quantitative determination of OSB (II) and coenz3 e A by HPLC showed that the activated OSB (II) was a monoester of coenzyme A. In addition, quantitative determination of OSB (II) by its known specific radioactivity and of coenzyme A by means of a coupled enzyme assay gave an identical result. Finally, when the -succinylbenzoate coenzyme A ligase preparation was incubated with C-OSB and H-coenzyme A, the ratio of... 

Recent calculations which take into account the corrected value of 0.320 for the Ei value of the DPN-DPNH couplet show that the AFJ for the hydrolysis of the pyrophosphate bond in ATP is —8,640 calories (Anfinsen and Kielley, personal communication). If this value is correct, the AFo for the hydrolysis of thiolesters, acetyl-P and N-acetyl imidazole will be about —8,600, —11,200 and —14,000 calories respectively instead of the higher values which are reported in this paper. 

Some evidence appears to support intramolecular general acid-specific base catalysis through electrostatic bonding as in 15139.141-143 recent worki , especially that on the relative order of hydrolysis, selenoester > thiolester > ester, suggests intramolecular nucleophilic catalysis through anchimeric assistance of the dimethylamino group as in 16 . 

Conventional structure-rate studies for the hydrolysis of thiolesters have been reported. ... 

Our assay for the detection of active acetate was based on the enzymic reaction with sulfanilamide. In the presence of the enzyme obtained from pigeon liver, the acetylation of sulfanilamide increased with increasing addition of active acetate . In our purification work we used, however, another, much less time-consuming assay which was based on the delayed color development as given by thiolesters with sodium nitro-prusside. When ammonium hydroxide is added to the mixture of thiolesters and nitroprusside no color is seen in the first moment. It only gradually develops in the following minutes due to the hydrolysis of the thiolester in the alkaline medium. The... 

Taking advantage of Lynen s spectrophotometric methods and the stability characteristics of thiolesters, it was readily shown that the incubation of phosphotransacetylase with acetyl-P and substrate levels of CoA led to the accumulation of a thiolester which was isolated and identified as acetyl-CoA.< > This represented the first net synthesis of acetyl-CoA in vitro and established once and for all the mechanism of the phosphotransacetylase catalyzed reaction. From equilibrium measurements of reaction (25), it was calculated that the standard free energy of hydrolysis of acetyl-CoA is about the same as that of the pyrophosphate bond of ATP (i.e. —8 kcal). The energy-rich nature of acetyl CoA was thus firmly established. 

Johnston (93,94) has studied the action of ciTstalline papain on thiolesters. The relative rates of hydrolysis at pH 5 of benzoylgly-... 

Hydrolysis kinetics for thiolesters deal with triphenylmethyl thiolben-zoates (AalI mechanism), hydrolysis and Ag -assisted n-butylaminoly-sis of ethyl thiolbenzoate, and thiolesters with an activating group attached to the carbonyl group. "... 

There are two forms of the hydrolytic enzyme, one of which is specific for short chain esters like retinyl acetate, even though this ester does not occur naturally The other has maximum activity with retinyl palmitate as substrate but also hydrolyses other long chain esters. As in the hydrolysis of cholesteryl esters, the enzyme is not just a non-specific esterase, but has quite definite specificity for retinyl esters. In vitamin A deficiency, the activity of the enzyme increases one hundred fold. The esterification enzyme resembles the low energy cholesteryl esterase in that neither ATP nor coenzyme A appear to take part in the reaction nor are free fatty acids or acyl-CoA thiolesters incorporated into retinyl esters. One of the major problems in this area of research is to identify the acyl donor, which may be, as in plasma cholesteryl ester biosynthesis, a phospholipid. 

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