LOCALIZED ELECTRON POLARIZATION IN A SUBSTRATE-ANALOG BINDING TO THE ACTIVE-SITE OF ENOYL-COA HYDRATASE - RAMAN-SPECTROSCOPIC AND CONFORMATIONAL-ANALYSES OF ROTAMERS OF HEXADIENOYL THIOLESTERS

The Raman bands associated with different rotamers of hexadienoyl ethy l thiolester, CH3-CH=CH-CH=CH-C(=O)-S-CH2-CH3, have been partially ass igned using recent conformational and vibrational spectroscopic analys es of ethyl thiocrotonate CH3-CH=CH-C(=O)-S-CH2-CH3.(1) In particular, rotational isomers involving the =CH-C(=O) and -S-CH2- axes have been characterized. The assignment of the vibrational modes of different c onformers of hexadienoyl ethyl thiolester was further facilitated by v ariable-temperature (+20 degrees C to -90 degrees C) Raman studies on the neat thiolester. High-quality Raman spectra of hexadienoyl-coenzym e A bound to the enzyme enoyl-CoA hydratase were obtained using 647.1- nm excitation and Raman difference spectroscopy. The findings provided by analysis of the ethyl thiolester model compound enable us to concl ude that conformational selection occurs for the CoA analog upon bindi ng, resulting in the presence of a single-rotamer population in the he xadienoyl moiety on the enzyme. The hexadienoyl-CoA was labeled with O -18 in the C=O group and, separately, with C-13 at the C2 position. A comparison of Raman data for the free and bound ligands, isotopically labeled and unlabeled, indicates that strong pi-electron polarization occurs in only a part of the hexadienoyl chain, viz, in the C=C-C=O fr agment, upon binding. The polarization gives rise to important contrib utions from canonical (resonance) forms of the type -C+-C=C-O-. In con trast, the C4=C5 linkage (where the carbon atom numbering is C6-C5=C4- C3=C2-C1=O) seems little perturbed in the bound ligand. The causes and mechanistic advantage of the observed localized polarization are disc ussed. (C) 1996 John Wiley & Sons.