Citric acid cycle enol intermediate

Steps 7-8 of Figure 29.12 Hydration and Oxidation The final two steps in the citric acid cycle are the conjugate nucleophilic addition of water to fumarate to yield (S)-malate (L-malate) and the oxidation of (S)-malate by NAD+ to give oxaloacetate. The addition is cataiyzed by fumarase and is mechanistically similar to the addition of water to ris-aconitate in step 2. The reaction occurs through an enolate-ion intermediate, which is protonated on the side opposite the OH, leading to a net anti addition. 

A similar aldol reaction is encountered in the Krebs cycle in the reaction of acetyl-CoA and oxaloacetic acid (see Section 15.3). This yields citric acid, and is catalysed by the enzyme citrate synthase. This intermediate provides the alternative terminology for the Krebs cycle, namely the citric acid cycle. The aldol reaction is easily rationalized, with acetyl-CoA providing an enolate anion nucleophile that adds to the carbonyl of oxaloacetic acid. We shall see later that esters and thioesters can also be converted into enolate anions (see Section 10.7). 

One of the simplest biochemical addition reactions is the hydration of carbon dioxide to form carbonic acid, which is released from the zinc-containing carbonic anhydrase (left, Fig. 13-1) as HC03-. Aconitase (center, Fig. 13-4) is shown here removing a water molecule from isocitrate, an intermediate compound in the citric acid cycle. The H20 that is removed will become bonded to an iron atom of the Fe4S4 cluster at the active site as indicated by the black H20. An enolate anion derived from acetyl-CoA adds to the carbonyl group of oxaloacetate to form citrate in the active site of citrate synthase (right, Fig. 13-9) to initiate the citric acid cycle. 

Fats and carbohydrates are metabolized down to carbon dioxide via an acetyl unit, CH3C=0, which is attached to a coenzyme, HSCoA, as a thioester called acetyl CoA. Acetyl CoA enters the citric acid cycle and eventually is converted to two molecules of carbon dioxide. The first step in the citric acid cycle is the aldol of acetyl CoA with oxaloacetate (Fig. 8.6). What is so elegant about this aldol is that the acidic and basic groups within the enzyme s active site provide a route that avoids any strongly acidic or basic intermediates. The enzyme accomplishes an aldol reaction at neutral pH, without an acidic protonated carbonyl or basic enolate intermediate via push-pull catalysis (Section 7.4.3). 

In the first reaction of the citric acid cycle, acetyl-CoA reacts with oxaloacetate to form citrate. The mechanism for the reaction shows that an aspartate side chain of the enzyme removes a proton from the a-carbon of acetyl-CoA, creating an enolate ion. This enolate ion adds to the keto carbonyl carbon of oxaloacetate and the carbonyl oxygen picks up a proton from a histidine side chain. This is similar to an aldol addition where the a-carbanion (enolate ion) of one molecule is the nucleophile and the carbonyl carbon of another is the electrophile (Section 18.10). The intermediate (a thioester) that results is hydrolyzed to citrate in a nucleophilic addition-elimination reaction (Section 16.9). 

Mevalonic acid itself is a product of acetate metabolism. Three molecules of acetate coenzyme A, produced by the citric acid cycle, are used to form mevalonic acid (Scheme 5.1). Two molecules undergo a Claisen condensation via acetyl-CoA-acetyltransferase enzyme [EC 2.3.1.9] to produce acetoacetyl-CoA, and a third is incorporated in a stereospecific aldol addition to the formation of p-hydroxy-p-methylglutaryl-CoA (HMG-CoA) by the aid of HMG-CoA synthase [EC 2.3.3.10]. The first Claisen reaction was found to involve formation of Cys-89 acetyl-5-enzyme reaction intermediate [9]. Then, Cys-378 residue on the active site of the enzyme activates a second molecule of acetyl-CoA to initiate the condensation reaction (Fig. 5.4) [11]. Similarly, in HMG-CoA synthases (S. aureus HMG-CoA synthase), Cysl 11/129 are the crucial residues of covalent attach to acetyl-CoA to produce acetyl-enzyme thioester with the subsequent loss of coenzyme A (Fig. 5.4). Glu79/95 residues are responsible for the enolization of acetyl-enzyme intermediate in order to react with acetoacetyl-CoA, which is bound to His233/264 residues [12]. 

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