Substrate Stereochemistry of the Enoyl‐CoA Hydratase Reaction

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Abstract
A specimen of stereospecifically 2‐tritiated 3‐hydroxybutyric acid was prepared by hydroboration of ethyl crotonate. It was assumed that the hydroboration reaction took asyncourse and hence that (2R,3S) + (2S,3R)‐3‐hydroxy[2‐3H1]butyric acid was formed after oxidation and hydrolysis. 3RS‐3‐Hydroxy[2‐3H2]butyric acid, symmetrically tritiated at C‐2, was prepared by isotopic exchange of ethyl acetoacetate in tritiated water, followed by reduction and hydrolysis. The 3R‐enantiomers of the acids listed under paragraphs (1) and (2) were destroyed enzymically by use of 3R‐specific 3‐hydroxybutyrate dehydrogenase and the residual 3S‐enantiomers were isolated. The resulting specimens of 2R,3S‐3‐hydroxy[2‐3H1]butyric acid and 3S‐3‐hydroxy[2‐3H2]‐butyric acid were converted chemically to the acyl‐CoA derivatives. These were incubated with enoyl‐CoA hydratase. In the presence of the enoyl‐CoA hydratase symmetrically labelled 3S‐3‐hydroxy[2‐3H2]butyryl‐CoA lost nearly 50% of its tritium label; 2R,3S‐3‐hydroxy[2‐3H1]butyryl‐CoA lost about 78%. It was concluded that the elimination of the elements of water from 3‐hydroxybutyryl‐CoA on the hydratase occurs stereospecifically withsyngeometry.