Molecular Mechanisms Of Fatty Acid Poxidation Enzyme Catalysis

Song-Yu Yang and Xue-Ying He Department of Pharmacology

New York State Institute for Basic Research in Developmental Disabilities

Staten Island, New York 10314

The P-oxidation of fatty acids is an important metabolic process that takes place in various organisms ranging from E. coli to Homo sapiens. In animal cells there are different P-oxidation systems in mitochondria and peroxisomes. Mitochondrial p-oxidation of fatty acids provides a significant part of the energy in some organs. For example, free fatty acids are the preferred substrate for energy production in heart. On the other hand, the peroxisomal P-oxidation system degrades very long-chain fatty acids and other uncommon carboxylic acids but does not provide useful energy.1 More complexity was found in plant cells where glyoxysomes—which correspond to peroxisomes—have a unique P-oxidation system.2 Fungi possess peroxisomalbifunctional P-oxidation enzymes employing D-specific substrates but not L-isomers.3 The stereochemical specificity of the latter enzymes is the opposite of that for the animal peroxisomal trifunctional enzyme.4

During prolonged fasting or long-lasting exercise, mitochondrial P-oxidation supplies energy to various tissues, producing acetyl-CoA for liver ketogenesis to supply circulating ketone bodies as an important alternative fuel in extrahepatic tissues. In addition to the mitochondrial matrix p-oxidation enzymes, a long-chain-specific P-oxidation enzyme system with a very long-chain acyl-CoA dehydrogenase and a trifunctional p~ oxidation complex has been discovered on the mitochondrial inner membrane.5

Although the fatty acid P-oxidation spiral comprises only four reactions, understanding of the complexity of fatty acid degradation has dramatically increased in recent years due to the discovery of a variety of new P-oxidation enzymes. This article will discuss the enzymes that catalyze the second and third steps of the P-oxidation pathway, an area of recent and substantial progress.


The second step of the P-oxidation spiral is the reversible hydration of 2-trans-enoyl-CoA to yield L-3-hydroxyacyl-CoA, catalyzed by enoyl-CoA hydratase.1 However, in fungi 2-trans-enoyl-CoA is hydrated by peroxisomal D-3-hydroxyacyl-CoA dehy-dratase to form D-3-hydroxyacyl-CoA.3 Enoyl-CoA hydratases are usually associated with the N-terminal region of multifunctional proteins except for the mitochondrial matrix enoyl-CoA hydratase and the E. coli long-chain enoyl-CoA hydratase10 (see Table 1). D-3-hydroxyacyl-CoA dehydratases are located on the C-terminal domain of the peroxisomal D-specific bifunctional P -oxidation enzyme3 or the central domain of 17(i-hydroxysteroid dehydrogenase type IV.8'9

An investigation of the stereochemistry of the reaction catalyzed by enoyl-CoA hydratase11 revealed that the P-addition/elimination reaction follows a syn course and the pro-R (but not pro-S) a-proton of L-3-hydroxyacyl-CoA is derived from water. On the basis of a double isotope effect study, Bahnson and Anderson12 have suggested that crotonase-catalyzed P-elimination is concerted. However, Gerlt and Gassman1314 have insisted that crotonase operates by a two-step process just as all the other enzymes that catalyze P-elimination reactions. They have argued that the crotonase active site provides an electrophile at the carbonyl oxygen of thioester group to form a short, strong hydrogen bond for enhancing the acidity of the a-proton of the substrate, so that in the first step a deprotonated catalytic residue abstracts the a-proton and then this protonated catalytic residue facilitates the departure of the P-leaving group in the second step. Despite lively controversy about the lifetime of the enolized intermediate, the advocates of the concerted mechanism12'15'16 and those of the stepwise mechanism13'14 all support the notion that a single general acid-base functional group is required for the catalysis of a P-addition/elimination reaction because of the reaction's syn stereochemical course.

Site-directed mutagenesis studies in our laboratory17 revealed that substitution of glutamine for glutamate-139 on the large subunit of the E. coli fatty acid oxidation complex caused a greater than 3,000-fold decrease in the kca, of enoyl-CoA hydratase without a significant change in the Km value. We identified the y- carboxyl group of glutamate-139 as the primary catalytic acid-base functional group of the E. coli crotonase in 1994. Meanwhile, Muller-Newen et al.18 found that a conserved glutamate-164 is the catalytic residue of rat liver mitochondrial enoyl-CoA hydratase. This glutamate residue is at position 135 of the mature enoyl-CoA hydratase, however, because a 29 residue leader sequence is cleaved when the precursor is transported into mitochondria; the catalytic residue of rat liver hydratase apparently corresponds to glutamate-139 of the E. coli crotonase (Figure 1).

Our studies on the pH dependence of kcJKm of the E. coli crotonase have revealed two pKa values (5.9 and 9.2).19 A pKa of 9.2 was assigned to glutamate-139, since a protonated catalytic residue is necessary for transferring a proton to the a-carbon of a,P-unsaturated fatty acyl-CoA thioesters. The results also suggested that crotonase possesses another catalytic residue involved in the hydration of 2-trans-enoyl-CoA. In order to identify the second catalytic residue, we mutated additional protic amino acid residues that are conserved in members of the enoyl-CoA hydratase family and investigated the catalytic properties of these mutant hydratases. We found that the substitution of glutamine for glutamate-119 on the large subunit of the E. coli fatty acid oxidation complex caused an 88-fold decrease in the catalytic rate with disappearance of a pKaof 5.9 of the wild type enoyl-CoA hydratase.19 The experimental data indicated that the y-carboxyl group of glutamate-119 serves as the second general acid-base functional group in cat alyzing the hydration of 2-trans-enoyl-CoA. According to the crystal structure of rat liver enoyl-CoA hydratase reported by Engel et al.,20 the 7-carboxyl group of glutamate-164 is close to oxygens of the 1-keto and 3-keto groups of acetoacetyl-CoA, an inhibitor bound to the hydratase active site, and the y-carboxyl group of glutamate-144 is hydrogen-bonded to the oxygen of the 3-keto group while the imino group of glycine-141 forms a short, strong hydrogen bond to the oxygen of the 1-keto group. It is noteworthy that glutamate-144 and glycine-141 are at positions 115 and 112, respectively, of the mitochondrial enoyl-CoA hydratase (see Fig. 1). The results of our site-directed mutagenesis studies prompted us to propose the catalytic mechanism of the E. coli enoyl-CoA hydratase. An outline of the hydratase reaction is shown in Fig. 2, and the same principles are applicable to the hydratases of other multifunctional p-oxidation enzymes and to monofunctional enoyl-CoA hydratases regardless of their substrate chain length specificities.

The molecular mechanism of the syn P -elimination reaction catalyzed by enoyl-CoA hydratase we proposed19 has a single concerted transition state where the general base catalyzes abstraction of the a-proton and the general acid catalyzes loss of the P-leaving group (Fig. 2). The harmonious cooperation of two general acid-base functional groups of hydratases could account for the observed high catalytic rate for the hydratase reaction, e. g., kca, ~ 775 s"1 reported for the hydration of crotonyl-CoA.17 It is not known why a single pKa of 8.5 was previously reported for rat liver enoyl-CoA hydratase.16 Nevertheless, the hypothesis that a single active-site base can mediate both proton transfers in a cyclic transition state12,16 and the view that the syn stereochemical course enables the hydration reaction to be catalyzed by a single group in a stepwise manner13,14 are no longer tenable. Other factors that could stabilize the transition state are included in the present model of hydratase catalysis (see Fig. 2). For example, hydrogen bonding of the imino group of glycine-116—which corresponds to glycine-141 of rat liver crotonase—with the carbonyl oxygen of the substrate could reduce the pKa of the a-proton,13,14 and its coordination with a nucleophile near the P-carbon could cause an electronic rearrangement in the acryloyl portion to enhance the electrophilicity of the P-carbon for substrate activation.15 The replacement of glycine-116 by phenylalanine would interfere the formation of such a hydrogen bonding, and did in fact result in inactivation of the crotonase.21

The syn p-addition/elimination of water proved not to be superior to an ami process in chemical efficiency.22 The reason why the reaction catalyzed by enoyl-CoA hydratases follows the syn stereochemical course is because the two catalytic residues of the hydratase are located on the same side of the substrate (see Fig. 2). The reaction mechanism of D-3-hydroxyacyl-CoA dehydratase catalysis is not known. If it follows the anti stereochemical course, the D-specific dehydratases of the peroxisomal multifunctional

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