Involvement of Glycine 141 in Substrate Activation by Enoyl-CoA Hydratase

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• 作者：Alasdair F. Bell ; Jiaquan Wu ; Yuguo Feng ; and Peter J. Tonge
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：February 13, 2001
• 年：2001
• 卷：40
• 期：6
• 页码：1725 - 1733
• 全文大小：104K
• 年卷期：v.40,no.6(February 13, 2001)
• ISSN：1520-4995

文摘

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 ahydrogen 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 theenone 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]. Inaddition, Raman spectra of HD-CoA bound to the G141P mutant enzyme demonstrate that the hydrogenbond 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 10⁶-fold decrease in k_cat and eliminates theability of the enzyme to polarize the substrate analogue. As G141 is part of a consensus sequence in theenoyl-CoA hydratase superfamily, the results presented here provide direct evidence for the importanceof the oxyanion hole in the reactions catalyzed by other family members.