Claisen enzymes carbanions

Kuo and Rose showed that the proton that is removed is retained by the enzyme (67). Stubbe and Abeles prepared an alternative substrate in which fluoride elimination competes with carboxylation 68, 69). Neither result defines the mechanism, but they do show that it is likely that the carbanion derived from the substrate is generated as an intermediate and therefore the reaction is not concerted. Definitive results come from double-isotope fraction studies by O Keefe and Knowles (70) and by Cleland and co-woricers (71). As described for Claisen enzymes, this methodology tests whether processes occur in one or two steps. Labeling of the carboxyl to be transferred with carbon-13 and the proton to be transferred as deuterium provided the means to do this test. The results indicate clearly that proton removal from the substrate to generate the carbanion and transfer of the carboxyl occurs in distinct steps. The resulting attack of the carb-... 

As we have seen in the section on thiolases, Claisen condensations normally involve activation of the electrophilic carhonyl through formation of a thioester and stabilization of the attacking carbanion also as a thioester. Dihydroxynaphthoyl-CoA synthase (MenB) catalyzes an intramolecular Claisen condensation reaction in which only the nucleophilic portion of the molecule has been converted to a thioester. This reaction is a component of the menaquinone biosynthetic pathway, and most studies have focused on the enzyme from M. tuberculosis based on the premise that this pathway may be a valid target for the development of novel compounds that inhibit both replicating and nonreplicating bacteria. ... 

Non-enzyme-catalysed bond-forming and -breaking reactions, such as the aldol and Claisen condensations, require strongly basic conditions to form the carbanion intermediates. In living systems, the carbanion formed in Claisen-type condensation is the a-anion of an acylthioester, usually acyl coenzyme A, rather than the usual acyl oxygen ester. 

In all the acetyl coenzyme-A-utilising enzymes which catalyse Claisen-type condensations the reaction involves the conversion of the acetyl methyl into a methylene group. A simple example illustrates the use of thiol esters both as carbanion-stabilis-ing systems and as readily hydrolysable esters. The conversion of glyoxalate to malate uses acetyl coenzyme. A probable mechanism is outlined below ... 

CHS (Fig. 2), the most studied member of the type III PKS family, is a ubiquitous enzyme in plants that catalyzes the first committed step in flavonoid biosynthesis, the elongation of the starter molecule 4-coumaroyl-CoA by addition of three acetate units derived from three molecules ofmalonyl-CoA [19]. After binding ofthe 4-coumaroyl moiety to the active site Cysl64, sequential polyketide chain elongation is initiated by the decarboxylation of malonyl-CoA to form an acetyl-CoA carbanion, followed by an intramolecular Claisen condensation step and subsequent cyclization and aromatization, yielding chalcone [20]. 

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