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?
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.
D'Ordine
R L
RL
Department of Chemistry, Brown University, Providence, Rhode Island 02912.
Bahnson
B J
BJ
Tonge
P J
PJ
Anderson
V E
VE
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D002417:Cattle; D004746:Enoyl-CoA Hydratase / Q000378:metabolism*; D004952:Esters / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D011522:Protons; D011812:Quinuclidines / Q000378:metabolism; D051381:Rats; D013237:Stereoisomerism; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D000214:Acyl Coenzyme A; D004952:Esters; D011522:Protons; D011812:Quinuclidines; D013438:Sulfhydryl Compounds; C090998:3-quinuclidinone; D004746:Enoyl-CoA Hydratase