Involvement of glycine 141 in substrate activation by enoyl-CoA hydratase

Raman spectroscopy has been used to investigate the structure of a substrat e analogue, hexadienoyl-CoA (HD-CoA), bound to wild-type enoyl-CoA hydratas e and G141P, a mutant in which a hydrogen bond to the substrate carbonyl ha s been removed. Raman spectra of isotopically labeled HD-CoAs, together wit h normal mode calculations, confirm the selective ground-state polarization of the enone fragment previously suggested to occur on binding to the wild type enzyme [Tonge, P. J., Anderson, V. E., Fausto, R., Kim, M., Pusztai-C arey, M., and Carey, P. R. (1995) Biospectroscopy 1,387-394]. In addition, Raman spectra of HD-CoA bound to the G141P mutant enzyme demonstrate that t he hydrogen bond between the G141 amide NH group and the substrate carbonyl is critical for polarization and activity. replacement of G141 with prolin e results in an approximately 10(6)-fold decrease in k(cat) and eliminates the ability of the enzyme to polarize the substrate analogue. As G141 is pa rt of a consensus sequence in the enoyl-CoA hydratase superfamily, the resu lts presented here provide direct evidence for the importance of the oxyani on hole in the reactions catalyzed by other family members.