Hydrolysis thioesters

FIGURE 20.5 Citrate is formed in the citrate synthase reaction from oxaloacetate and acetyl-CoA. The mechanism involves nncieophiiic attack by the carbanion of acetyl-CoA on the carbonyl carbon of oxaloacetate, followed by thioester hydrolysis. 

The efficiency of the intein cleavage varies with the type of intein used for fusion and with the cleavage conditions (pH, temperature). Since usage of different thiol compounds results not only in a diverse splicing activity but also in a diverse rate of thioester hydrolysis, both parameters have to be taken into account. Specific protein properties, like the structure or the sequence at the C-terminus of the target protein, may also affect the cleavage efficiency (28,29). 

Intermediate 113 was transformed into the trimethylsilyl methyl ketone 119 in quantitative yield via the acid chloride, followed by the addition of trimethylsilylmethyl lithium (Scheme 2.13). Subsequent Peterson olefination of aldehyde 108 with 119 resulted in the required unsaturated ketone 120 in 95% yield. Subsequent desilylation and thioester hydrolysis afforded a 97% of the seco acid 121. Lactonization was achieved in 32% yield via the phosphoric acid-mixed anhydride, however this procedure also formed about 25% of the dimeric bis lactone. Following removal of the acetonide under the action of acid, a nonselective oxidation with RuCl2(Ph3P)3 produced a 1 1 mixture of the natural... 

Chiral inversion of the 2-APAS is presented in detail as a whole chapter (Chap. 8) in this book. However, a brief overview of its mechanism is presented here. The mechanism of the inversion reaction (Fig. 14) has been extensively investigated both in vitro and in vivo and involves stereoselective formation of the acyl-coenzyme A (CoA) thioester of the (i )-2-APA, via an adenosine monophosphate intermediate. Once formed, the (i )-profenyl-CoA thioester undergoes epimerization at the center of chirality in the profen moiety, which is thought to proceed via an enolate intermediate, to yield the corresponding (5)-profenyl-CoA thioester. Hydrolysis of the acyl-CoA thioesters results in the liberation of the free acid. The enzyme... 

The second enzyme of the glyoxylase system, the thioesterase, is specific for thioesters of glutathione and its analogues. Thioester hydrolysis and transacylation will be discussed in subsequent sections. 

Figure 8.P31 gives the pH-rate profile for hydrolysis of thioesters A-D and shows a dependence on the nature of the substituents in the alkylthio group. Propose a mechanism which would account for the pH-rate profile of each compound. 

Fig. 8.P3I. Plot of the pseudo-first-order rate constants for hydrolysis of thioesters A (O), B ( ), C (A), D (A) as a fiinction of pH at 50°C and ionic strength 0.1 (KCI). Lines are from fits of the data to = kon(K /H+)) + (k KJK + [//+])) where koH is the hydroxide term and is the intramolecular assistance term for B and C and from linear regression for A and D. Reproduced from problem reference 31 by permission of the American Chemical Society.

The activation of acyl groups for transfer by CoA can be appreciated by comparing the hydrolysis of the thioester bond of acetyl-CoA with hydrolysis of a simple oxygen ester ... 

The overall result is a "hydrolysis" of the thioester group without involvement of water. 

In contrast to oxoesters, the a-protons of thioesters are sufficiently acidic to permit continuous racemization of the substrate by base-catalyzed deprotonation at the a-carbon. Drueckhammer et al. first demonstrated the feasibility of this approach by performing DKR of a propionate thioester bearing a phenylthiogroup, which also contributes to the acidity of the a-proton (Figure 4.14) [39a]. The enzymatic hydrolysis of the thioester was coupled with a racemization catalyzed by trioctylamine. Owing to the insolubility of the substrate and base in water, they employed a biphasic system (toluene/H2O). Using P. cepacia (Amano PS-30) as the enzyme and a catalytic amount of trioctylamine, they obtained a quantitative yield of the corresponding... 

Figure29-1. Partial reactions in the attachment of ubiquitin (UB) to proteins. (1) The terminal COOH of ubiquitin forms a thioester bond with an -SH of E, in a reaction driven by conversion of ATP to AMP and PP. Subsequent hydrolysis of PP by pyrophosphatase ensures that reaction 1 will proceed readily. (2) A thioester exchange reaction transfers activated ubiquitin to Ej. (3) E3 catalyzes transfer of ubiquitin to e-amino groups of lysyl residues of target proteins.

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