Enzyme enoyl-CoA hydratase

The oxidation of fatty acids within the Knoop-Lynen cycle occurs in the matrix. The Knoop-Lynen cycle includes four enzymes that act successively on acetyl-CoA. These are acyl-CoA dehydrogenase (FAD-dependent enzyme), enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase (NAD-dependent enzyme), and acetyl-CoA acyltrans-ferase. Each turn, or revolution, of the fatty acid spiral produces... 

Figure 7.15 The interaction between valproate and the mitochondrial p-oxidation system. Dark arrows denote depletion. Filled in circle is the carnitine transporter. 2,4, VPA 2,4, diene-VPA CoA ester. This reactive metabolite damages the enzyme enoyl CoA hydratase and the mitochondrial membranes and depletes GSH, as indicated.

Mitochondria contain three acyl CoA dehydrogenases which act on short-, medium- and long-chain acyl CoAs, respectively. In contrast, there is just one each of the enzymes enoyl CoA hydratase, hydroxyacyl CoA dehydrogenase and (3-ketothiolase which all have a broad specificity with respect to the length of the acyl chain. 

Conjugate addition of water. The a,/3-unsaturated acyl CoA produced in step 1 reacts with water by a conjugate addition pathway (Section 19.14) to jneld a /3-hydroxy acyl CoA in a process catalyzed by the enzyme enoyl CoA hydratase. Water as nucleophile adds to the /3 carbon of the double bond, yielding an enolate ion intermediate, which is then protonated to yield an alcohol. 

H20 adds across the double bond, and a p-hydroxyacyl CoA is formed. —Enzyme enoyl CoA hydratase... 

Willadsen and Eggerer (75) have studied the stereochemistry of the enzyme acetyl CoA acetyltransferase, a key enzyme in both the terminal step in C-3 oxidation of fatty acids and the initial step in the biosynthesis of terpenes and steroids. The enzyme, when incubated separately with (2S)-[2-2Hi,2-3Hi]aceto-acetyl CoA and the (2R) isomer gave two moles of acetyl CoA as depicted in Scheme 17. Eggerer et al. (76) utilized the enzyme enoyl CoA hydratase to convert properly labeled crotonyl CoA, via syn addition, to the doubly isotopically labeled 3-hydroxyacyl CoA derivatives needed in this study. A discussion of this unique type of hydration has been presented by Rose (9). The labeled... 

FIGURE 24.15 The conversion of trans- and m-enoyl CoA derivatives to l- and d-/3-hydroxyacyl CoA, respectively. These reactions are catalyzed by enoyl-CoA hydratases (also called crotonases), enzymes that vary in their acyl-chain length specificity. A recently discovered enzyme converts ram-enoyl-CoA directly to D-/3-hydroxyacyl-CoA. 

Polyunsaturated fatty acids pose a slightly more complicated situation for the cell. Consider, for example, the case of linoleic acid shown in Figure 24.24. As with oleic acid, /3-oxidation proceeds through three cycles, and enoyl-CoA isomerase converts the cA-A double bond to a trans-b double bond to permit one more round of /3-oxidation. What results this time, however, is a cA-A enoyl-CoA, which is converted normally by acyl-CoA dehydrogenase to a trans-b, cis-b species. This, however, is a poor substrate for the enoyl-CoA hydratase. This problem is solved by 2,4-dienoyl-CoA reductase, the product of which depends on the organism. The mammalian form of this enzyme produces a trans-b enoyl product, as shown in Figure 24.24, which can be converted by an enoyl-CoA isomerase to the trans-b enoyl-CoA, which can then proceed normally through the /3-oxidation pathway. Escherichia coli possesses a... 

Enzymes 7,9, and 13 form a trifunctional protein associated with the inner face of the inner mitochondrial membrane. Very-long-chain acyl-CoA dehydrogenase is also associated with other inner mitochondrial membranes while the other enzymes are in the matrix and may be loosely associated with the inner face of the inner membrane. A medium-chain 2-enoyl-CoA hydratase may also be present in the mitochondrial matrix. 

The metabolism of ferulate to vanillin by Pseudomonas fluorescens strain AN103 is carried out by an enoyl-SCoA hydratase/isomerase rather than by oxidation, and the enzyme belongs to the enoyl-CoA hydratase superfamily (Gasson et al. 1998). 

The chain shortening pathway has not been characterized in detail at the enzymatic level in insects. It presumably is similar to the characterized pathway as it occurs in vertebrates. These enzymes are a partial P-oxidation pathway located in peroxisomes [29]. The key enzymes involved are an acyl-CoA oxidase (a multifunctional protein containing enoyl-CoA hydratase and 3-hy-droxyacyl-CoA dehydrogenase activities) and a 3-oxoacyl-CoA thiolase [30]. These enzymes act in concert to chain shorten acyl-CoAs by removing an acetyl group. A considerable amount of evidence in a number of moths has accumulated to indicate that limited chain shortening occurs in a variety of pheromone biosynthetic pathways. 

Bifunctional protein deficiency. The enzyme defect involves the D-bifunctional protein. This enzyme contains two catalytic sites, one with enoyl-CoA hydratase activity, the other with 3-hydroxyacyl-CoA activity [13]. Defects may involve both catalytic sites or each separately. The severity of clinical manifestations varies from that of a very severe disorder that resembles Zellweger s syndrome clinically and pathologically, to somewhat milder forms. Table 41-6 shows that biochemical abnormalities involve straight chain, branched chain fatty acids and bile acids. Bifunctional deficiency is often misdiagnosed as Zellweger s syndrome. Approximately 15% of patients initially thought to have a PBD have D-bifunctional enzyme deficiency. Differential diagnosis is achieved by the biochemical studies listed in Table 41-7 and by mutation analysis. 

Yang and Schulz also formulated a treatment of coupled enzyme reaction kinetics that does not assume an irreversible first reaction. The validity of their theory is confirmed by a model system consisting of enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) with 2,4-decadienoyl coenzyme A as a substrate. Unlike the conventional theory, their approach was found to be indispensible for coupled enzyme systems characterized by a first reaction with a small equilibrium constant and/or wherein the coupling enzyme concentration is higher than that of the intermediate. Equations based on their theory can allow one to calculate steady-state velocities of coupled enzyme reactions and to predict the time course of coupled enzyme reactions during the pre-steady state. 

The last three steps of this four-step sequence are catalyzed by either of two sets of enzymes, with the enzymes employed depending on the length of the fatty acyl chain. For fatty acyl chains of 12 or more carbons, the reactions are catalyzed by a multienzyme complex associated with the inner mitochondrial membrane, the trifunctional protein (TFP). TFP is a heterooctamer of 4/34 subunits. Each a subunit contains two activities, the enoyl-CoA hydratase and the /3-hydroxyacyl-CoA dehydrogenase the /3 subunits contain the thiolase activity. This tight association of three enzymes may allow efficient substrate channeling from one active site to the... 

Figure 11 The putative catabolic pathway of L-leucine and its implications for strain improvement. For a promising host strain, the pathway to be blocked is indicated with thick double lines and the pathways to be fortified are indicated with thick arrows. Abbreviations for enzymes participating in the L-leucine catabolism and the acylation of tylosin VDH, valine (branched-chain amino acid) dehydrogenase BCDFI, branched-chain a-keto acid dehydrogenase IVD (AcdH), isovaleryl-CoA dehydrogenase (acyl-CoA dehydrogenase) MCC, 3-methylcrotonyl-CoA carboxylase EH, enoyl-CoA hydratase AcyA, mac-rolide 3-O-acyltransferase AcyBl, macrolide 4"-(9-acyltransferase.

Fig. 2. Metabolic pathways for PHA biosyntheis in fad mutant E. coli strains used in this study. Enoyl-CoA hydratase, epimerase, and 3-ketoacyl-CoA or ACP reductase have been suggested to supply PHA precursors from inhibited b-oxidation pathway. The crosses indicate inactivation of corresponding enzymes. The question mark represents uncharacterized enzyme. Enzymes involved in the metabolic pathways shown have been described previously FabG (21,32), YfcX (24,33), MaoC (34), PhaA (36), and PhaB (36).

Enoyl-CoA hydratase catalyses the hydration of unsaturated acyl-CoA. This enzyme has broad specificity and can act on a-, P- (or A -) unsaturated CoA in trans or cis configuration. The products formed are... 

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