ELECTRONIC REARRANGEMENT INDUCED BY SUBSTRATE-ANALOG BINDING TO THE ENOYL-COA HYDRATASE ACTIVE-SITE - EVIDENCE FOR SUBSTRATE ACTIVATION

A series of alpha,beta unsaturated CoA thiol esters have been characte rized spectroscopically when they form noncovalent complexes at the ac tive site of enoyl-CoA hydratase. The UV spectra of all of the thiol e sters display significant red shifts when the esters are bound to the crotonase active site. The red shift increases with the ability of a p ara substituent of substituted cinnamoyl-CoA thiol esters to donate el ectrons by resonance. The affinity of the substituted cinnamoyl-CoA th iol esters is enhanced by electron-donating substituents, with the slo pe of the log of the ratio of the inhibition constants versus sigma(p) (+) being near unity. Affinity is also increased by either para or met a electron-withdrawing substituents, suggesting that the enzyme stabil izes a partial positive charge at C-3. Binding to crotonase was shown to decrease the shielding of [3-C-13,3-H-2] cinnamoyl-CoA by +3.2 ppm, consistent with an increased partial positive charge at C-3. The Rama n spectra of cinnamoyl-CoA bound at the crotonase active site similarl y reflect the significant electronic ground state changes in the pi el ectronic structure of the bound substrate. These data show that a majo r rearrangement of electrons occurs in the acryloyl portion of the cin namoyl group upon binding, while only a minor perturbation occurs to t he distribution of electrons in the phenyl ring. The strong electron p olarization seen localized in the acryloyl moiety is consistent with a model in which the active site provides an electrophile at the C=O ox ygen (e.g., in the form of a positive charge or strong H-bonds) and a nucleophile near the C-3 carbon of the cinnamoyl moiety.