Enolic Intermediates in Enzymatic Reactions

The high values of the pK s of carbon acid substrates and the associated instability of enolate anion intermediates in nonenzymatic reactions first led to the expectation that these intermediates could not be rendered kinetically competent in enzymatic reactions [3]. As a result, the expectation was that these reactions must be concerted, thereby avoiding the problem of how an active site might provide sufficient, significant stabilization of the intermediates. However, the weight of the experimental evidence now is that enzymes that abstract protons from carbon acids are able to sufficiently stabilize enolate anion intermediates so that they can be kinetically competent. 

Citrate synthase catalyzes the metabolically important formation of citrate from ace-tyl-CoA and oxaloacetate [68]. Asp-375 (numbering for pig CS) has been shown to be the base for the rate-limiting deprotonation of acetyl-CoA (Fig. 5) [69]. An intennediate (which subsequently attacks the second substrate, oxaloacetate) is believed to be formed in this step the intermediate is thought to be stabilized by a hydrogen bond with His-274. It is uncertain from the experimental data whether this intermediate is the enolate or enol of acetyl-CoA related questions arise in several similar enzymatic reactions such as that catalyzed by triosephosphate isomerase. From the relative pK values of Asp-375... 

The decarboxylation reaction usually proceeds from the dissociated form of a carboxyl group. As a result, the primary reaction intermediate is more or less a carbanion-like species. In one case, the carbanion is stabilized by the adjacent carbonyl group to form an enolate intermediate as seen in the case of decarboxylation of malonic acid and tropic acid derivatives. In the other case, the anion is stabilized by the aid of the thiazolium ring of TPP. This is the case of transketolases. The formation of carbanion equivalents is essentially important in the synthetic chemistry no matter what methods one takes, i.e., enzymatic or ordinary chemical. They undergo C—C bond-forming reactions with carbonyl compounds as well as a number of reactions with electrophiles, such as protonation, Michael-type addition, substitution with pyrophosphate and halides and so on. In this context,... 

AcetoacetylCoA thiolase (E.C. 2.3.1.9), acetoacetylCoA reductase (E.C. 1.1.1.36), and polyhydroxybutyrate synthetase12471 are the enzymes involved in polyester synthesis. AcetoacetylCoA thiolase catalyzes the head-to-tail Claisen condensation of two acetylCoA molecules. In this reaction, the active site cysteine attacks acetylCoA to form a thioester enzyme intermediate, which then reacts with the enolate derived from enzymatic deprotonation of the other acetylCoA. Mechanistic studies have been performed on this enzyme from Zooglea ramigera, which has been cloned and overexpressed12471. It has been established that the thiolase will form acyl enzyme intermediates with a number of acylCoA substrates, but will only accept acetylCoA as the nucleophile. After subsequent reduction, this results in all polymer units possessing a P-hydroxy group. These polymers are also useful sources of (R)-P-hydroxy acids[2481. 

This and other information show that nine Cg units from malonyl-coenzyme A and one C3 unit from propionyl-coenzyme A condense to form the linear polyketide intermediate shown below. These units are joined by acylation reactions that are the biosynthetic equivalent of the malonic ester synthesis we studied in Section 18.7. These reactions are also similar to the acylation steps we saw in fatty acid biosynthesis (Special Topic E in WileyPLUS). Once formed, the linear polyketide cyclizes by enzymatic reactions akin to intramolecular aldol additions and dehydrations (Section 19.6). These steps form the tetracyclic core of akiavinone. Phenolic hydroxyl groups in akiavinone arise by enolization of ketone carbonyl groups present after the aldol condensation steps. Several other transformations ultimately lead to daunomycin ... 

This enzyme catalyses the decarboxylation of the ) -ketoacid oxaloacetate, with the same stoichiometry as acetoacetate decarboxylase. The former however, requires a Mn ion for activity and is insensitive to the action of sodium borohydride. This duality of mechanism is not unlike the one observed for enzymatic aldol condensation, where enzymes of Class 1 react by forming Schiff-base intermediates, whereas enzymes of Class II show metal ion requirements [47]. Oxaloacetate decarboxylase from cod also catalyses the reduction by borohydride of the enzymatic reaction product pyruvate. This is evidenced by the accumulation of D-lactate in presence of enzyme, reducing agent, and manganous ions. It has been proposed that both reduction and decarboxylation occur by way of an enzyme-metal ion-substrate complex in which the metal ion acts as an electron sink, thereby stabilizing the enolate ion formed in the decarboxylation reaction [48] ... 

In 1996, the first successful combination of an enzymatic with a nonenzymatic transformation within a domino process was reported by Waldmann and coworkers [6]. These authors described a reaction in which functionalized bicy-clo[2.2.2]octenediones were produced by a tyrosinase (from Agaricus bisporus) -catalyzed oxidation of para-substituted phenols, followed by a Diels-Alder reaction with an alkene or enol ether as dienophile. Hence, treatment of phenols such as 8-1 and an electron-rich alkene 8-4 in chloroform with tyrosinase in the presence of oxygen led to the bicyclic cycloadducts 8-5 and 8-6 in moderate to good yield (Scheme 8.1). It can be assumed that, in the first step, the phenol 8-1 is hydroxylated by tyrosinase, generating the catechol intermediate 8-2, which is then again oxidized enzy-... 

Such a condensation is mediated by the enzyme 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS). The mechanism of this catalysis is outlined in Figure 1.21. An initial frani-thioesterase step transfers the acetyl group of the first acetyl-CoA to an enzymatic cysteine. In the Claisen condensation phase of the reaction, the a-carbon of a second acetyl-CoA is deprotonated, forming an enolate. The enolate carbon attacks the electrophilic thioester carbon, forming a tetrahedral intermediate which quickly collapses to expel the cysteine thiol [22]. 

Although aminocatalysis of the aldol reaction via enamine intermediates is an important enzymatic strategy and several bioorganic studies of the subject have appeared, applications in preparative organic synthesis, particularly in intermolecular aldol addition reactions, have been published only sporadically. Despite the often-used Mukaiyama-aldol reaction of enol ethers and Stork s vell-developed enamine chemistry [37, 38], aldolizations of preformed enamines are rare. One report describes Le vis acid-catalyzed aldolizations of preformed enamines vith aldehydes that furnish aldol addition products [39]. Aldol condensation reactions of preformed enamines vith aldehydes have also been described [40]. Only enamine-catalytic aldolizations, vhich are primary and secondary amine-catalyzed aldol reactions, vill be discussed in this chapter, ho vever. 

Alone among the characterized members of the superfamily, the cMyc promoter binding protein (MBPI) has no known enzymatic function, In addition to MR and MLE, the superfamily is now known to include enola,se, which has also been structurally and mechanistically characterized. Like MR and MLE, the enolase reaction is initiated by abstraction of a proton a to a carboxylate group (equation I), followed by formation of a stabilized enolate anion intermediate. 

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