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

Biochemistry
A F BellP J Tonge

Abstract

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.

References

May 1, 1959·Archives of Biochemistry and Biophysics·G L ELLMAN

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Citations

Dec 31, 2002·Current Opinion in Structural Biology·Jung-Ja P Kim, Kevin P Battaile
Mar 25, 2009·Journal of the American Chemical Society·M Ashley SpiesJerome Baudry
Feb 11, 2011·Chemistry : a European Journal·Imre PápaiRik K Wierenga
May 14, 2003·The Journal of Biological Chemistry·Ila MisraHenry M Miziorko
Dec 7, 2002·Bioorganic & Medicinal Chemistry·Gautam Agnihotri, Hung-wen Liu

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