Enoyl-Coenzyme A Hydratase-Catalyzed Exchange of the .alpha.-Protons of Coenzyme A Thiol Esters: A Model for an Enolized Intermediate in the Enzyme-Catalyzed Elimination?

Abstract

3-Quinuclidinone catalyzes the exchange of the alpha-protons of butyryl-coenzyme A (CoA) with a second-order rate constant of 2.4 x 10(-6) M-1 s-1. In contrast, enoyl-CoA hydratase catalyzes the stereospecific exchange of the pro-2S proton of butyryl-CoA with a maximum second-order rate constant of ca. 8 x 10(2) M-1 s-1. This isotope exchange reaction is completely stereospecific within the limits of experimental detection (over 600-fold). The enzyme-catalyzed exchange is dependent on pD, decreasing above a pKa of 8.8 and below a pKa of 8.1, but independent of the buffer concentration. The stereospecificity of the exchange was unexpected because the pro-2R hydrogen is abstracted during the enzyme-catalyzed dehydration of 3(S)-hydroxybutyryl-CoA. In spite of the ability to exchange the pro-2S hydrogen, the stereospecificity of the dehydration reaction was determined to be better than 1 in 10(5) as no incorporation of 2H into the alpha-position of crotonyl-CoA or into the pro-2S position of 3(S)-hydroxybutyryl-CoA was detected during prolonged equilibrations with enoyl-CoA hydratase. Both the exchange of the alpha-proton and the dehydration activity of the enzyme are diminished by over 100-fold in a site-directed mutation of rat liver enoyl-CoA hydratase, where glutamate-164 is changed to glutamine, strongly suggesting that the same active site base is responsible for proton abstraction in both the dehydration and solvent exchange reactions. The enoyl-CoA hydratase-catalyzed exchange of the alpha-protons becomes nonstereospecific when the acidity of the alpha-protons is enhanced. While alpha-proton abstraction can be observed when no elimination reaction is possible, there is no evidence for proton abstraction without elimination in the crotonase equilibrations with 3(S)-hydroxybutyryl-CoA, 3-hydroxypropionyl-CoA, or 3-chloropropionyl-CoA. The differences in the isotope exchange and dehydration reactions emphasize the importance of the 3-hydroxyl group in promoting elimination and are consistent with a concerted elimination mechanism.

Protonic Equilibria in the Reductive Half-Reaction of the Medium-Chain Acyl-CoA Dehydrogenase

“…In particular, the polarization seen in this work shows striking parallels with that observed for the binding of cinnamoyl-CoA analogues to enoyl-CoA hydratase (34,59) and benzoyl-CoA derivatives to 4-chlorobenzoyl-CoA dehalogenase (60,61). Strong, ground-state polarization of the carbonyl was observed via Raman and 13 C NMR with accompanying sizable red shifts in the UV/VIS spectrum of the bound ligands.…”

“…Further, the pH dependence of the polarization observed with enoyl-CoA hydratase disappears upon mutation of the active site base, Glu164, to a Gln residue (59). Loss of polarization on deprotonation of Glu164 in the hydratase might involve unfavorable electrostatic repulsion between the polarized thioester carbonyl group and the carboxylate of Glu164 (34,59). In addition to these local effects, the equilibrium between species 3 and 5 in the acyl-CoA dehydrogenases (Scheme 5) would also result in modulation of polarization.…”

Crystal Structure of Mycobacterium tuberculosis MenB, a Key Enzyme in Vitamin K2 Biosynthesis

“…This is significant since Glu-164 is a key catalytic residue in the reaction catalyzed by enoyl-CoA hydratase (68). While it is intriguing to speculate that Asp-185 in MenB plays a similar role to that of Glu-164, we note that Asp-185 is not conserved in the MenB family, and is sometimes replaced by a glycine (Fig.…”

Metabolism of Ferulic Acid to Vanillin

“…The equivalent region of the derived amino acid sequence of ORF A stretches from Gly 114 to Met 172 ; within this region, the derived sequences of ORF A and ORF2 are almost identical, differing in only one residue, at Gln 166 . It is known from site-directed mutagenesis studies that a conserved Glu residue functions as a catalytic base in the reactions catalyzed by mitochondrial enoyl-SCoA hydratase and mitochondrial ⌬ 3 -cis-⌬ 2 -trans-enoyl-SCoA isomerase (9,32,33) and by the enoyl-SCoA hydratase activity of the multifunctional fatty acid oxidation large ␣-subunit of E. coli (34). This residue appears conserved in all members of the superfamily with enoyl-SCoA hydratase or ⌬ 3 -cis-⌬ 2 -trans-enoyl-SCoA isomerase activity, and it is present in the derived amino acid sequences of ORF A and ORF2, at residue 143 and 111, respectively.…”