Thioesterification

Selective thioesterification of glyceric acid and lactic acid.[187]... 

CY Shiau, MF Byford, RT Aplin, JE Baldwin, CJ Schofield. L-Delta-(alpha-amino-adipoyl)-L-cysteinyl-D-valine synthetase thioesterification of valine is not obligatory for peptide bond formation. Biochemistry 36 8798-8806, 1997. 

A second straightforward thioesterification method employs simple condensation conditions of the C-terminal carboxylic acid and thiols by using benzotriazol-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) or other condensation... 

SCHEME 113 Synthesis of glycopeptide-athioesters by (a) using safety-catch linker and (b) thioesterification of activated C-terminal carboxylic acids. Boc, ferf-butoxycarbonyl. 

In addition to thioesterifications employing the C-terminal condensation with alkyl-thiols, the Danishefsky group demonstrated an alternative type of thioesterification (Scheme 11.6) [74], A suitably side chain-protected peptide acid is condensed with an amino acid 2-dithioethyl phenyl ester. The 2-dithioethyl phenyl ester is prone to rearrangement to a thioester through O S transesterification under reducing (NCL) conditions. 

SCHEME 11.6 Glycopeptide thioesterification via O-to-S acyl shift. MESNa, sodium 2-mercaptoethanesulfonate. 

Fig. 2. Scheme of protein splieing. Cleavage pathway proposed for intein that possesses a cysteine residue in eaeh spliee junetion. In the initial step a linear thioester intermediate is formed by an N-S acyl rearrangement at Cysi (N-terminal amino acid of the intein). Next, traw -thioesterification that involves nucleophilic attack of the side-... 

Fig. 4. Scheme of NCL. The mechanism allows the straightforward preparation of small proteins with native backbone structures from fully unprotected synthetic peptide building blocks. The initial tran -thioesterification step includes the chemo-selective reaction between one peptide with a C-terminal a-thioester group (peptide 1) and second peptide with an N-terminal cysteine residue (peptide 2). Generated thio-ester-linked intermediate spontaneously rearranges to form a native peptide bond at the site of ligation. 

An alternative elegant native ligation approach has recently been proposed where the Staudinger reaction is exploited to couple the carboxy component as a thioester via trans-thioesterification to a suitable phosphine scaffold that reacts with azidoacyl peptides to produce the imino-X, -phosphine. This chemical ligation is followed by nucleophilic attack by the imino-X -phosphine nitrogen on the thioester to form the amide bondt °°l (Section 2.2.1.10). 

Laposata M., and Muszbek L. (1996). Thioesterification of platelet proteins with saturated and polyunsaturated fatty acids. Lipids. 31 Suppl, S217-S221. 

Two subunit FASs are also present in the palmitate synthase from Saccharomyces cerevisiae. These proteins form a multi-subunit complex, 06 6 and share active sites across the two subunits. The FAS proteins involved in primary metabolism in A. nidulans are shorter than HexA and HexB, used for formation of the hexanoyl CoA starter unit. Recent evidence suggests that the PKS is part of this protein complex (Figure 4B). The FAS/PKS complex (also called the NorS complex) has the stoichiometry a2P2Y2- Presumably hexanoylCoA never becomes a free unit and is transferred directly to the PKS by an internal trans thioesterification process. This explains why added precursor hexanoylCoA feeds poorly into a strain of Aspergillus in which HexA was inactivated. Furthermore, release of hexanoylCoA from the complex has not been found. 

In normal myocardium, the first step in fatty acid utilization is thioesterification catalyzed by acylCoA synthetase. There are three potential metabolic fates of the synthesized acylCoA including 1) transport into the mitochondrial matrix for subsequent P-oxidation, 2) utilization as an intermediate in polar and nonpolar lipid synthesis, and 3) hydrolysis by acylCoA hydrolase (i.e., a net futile cycle). In normal myocardium, the major fraction of synthesized acylCoA is transported into the mitochondrial matrix by sequential transesterification reactions catalyzed by carnitine acyltransferase. AcylCoA in the mitochondrial matrix space is sequentially oxidized in two-carbon units to produce acetylCoA, which is accompanied by the production of the reducing equivalents, NADH and FADH2. 

The first reaction in the fatty acid oxidation sequence is the thioesterification of the acid by CoA-SH. This involves bond formation between the CoA-S and the fatty acid and is therefore endergonic it is achieved by linking it to the simultaneous hydrolysis of ATP to AMP. The enzyme which does the job is a thiokinase (27). 

Thioesterification takes place within the cytoplasm but the rest of the reactions which constitute /5-oxidation occur within the mitochondria. However, fatty acyl CoA cannot penetrate the mitochondrial membrane, so for the transport to occur, the CoA group is substituted by a compound called carnitine, which carries the fatty acyl group into the mitochondria. Once inside it reverts to acyl CoA. 

Figure 22 The two half-reactions catalyzed by 4-coumarate CoA ligase (4CL). (a) Adenylation and (b) thioesterification.

These enzymes, like 4CL, share a common reaction mechanism for formation of adenylate intermediates, with the catalytic processes of adenylation half-reaction and thioesterification half-reaction occurring via a ping-pong mechanism. 

Figure 24 The proposed roles of key catalytic amino acid residues in (a) adenylation and/or (b) thioesterification during 4-chlorobenzoate CoA (CBL) catalysis.

Based on the structure of M. tuberculosis LipB as well as prior mechanistic studies, a mechanism for catalysis by LipB was proposed (Figure 14). The reaction is initiated by binding of octanoyl-ACP to LipB, upon which rt7 j--thioesterification takes place to generate the covalent octanoyl-LipB intermediate. The appropriate LCP (denoted E2) binds subsequent to release of holo-ACP, whereupon amide formation takes place to afford the corresponding octanoyl-LCP. It is postulated that proton transfers to and from substrates and products are mediated, at least in part, by Lysl42. ... 

Fig. 10.2-6 Auxiliary mediated native chemical ligation, (a) trans thioesterification, S-to-acyl tranfer, removal of auxiliary, (b) Tmb auxiliary (c) Z-phenylethane thiol auxiliary.

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