Involvement of glycine 141 in substrate activation by enoyl-CoA hydratase.
Raman spectroscopy has been used to investigate the structure of a substrate analogue, hexadienoyl-CoA (HD-CoA), bound to wild-type enoyl-CoA hydratase and G141P, a mutant in which a hydrogen bond to the substrate carbonyl has been removed. Raman spectra of isotopically labeled HD-CoAs, together with 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-Carey, 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 the hydrogen bond between the G141 amide NH group and the substrate carbonyl is critical for polarization and activity. Replacement of G141 with proline 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 part of a consensus sequence in the enoyl-CoA hydratase superfamily, the results presented here provide direct evidence for the importance of the oxyanion hole in the reactions catalyzed by other family members.
Bell
A F
AF
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794-3400, USA.
Wu
J
J
Feng
Y
Y
Tonge
P J
PJ
D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002247:Carbon Isotopes / Q000378:metabolism; D004746:Enoyl-CoA Hydratase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D005998:Glycine / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D010103:Oxygen Isotopes / Q000378:metabolism; D011392:Proline / Q000235:genetics; D011487:Protein Conformation; D051381:Rats; D012996:Solutions; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity / Q000235:genetics
D000214:Acyl Coenzyme A; D002247:Carbon Isotopes; D010103:Oxygen Isotopes; D012996:Solutions; C417225:hexadienoyl-coenzyme A; D011392:Proline; D004746:Enoyl-CoA Hydratase; D005998:Glycine