Evidence from Raman Spectroscopy That InhA, the Mycobacterial Enoyl Reductase, Modulates the Conformation of the NADH Cofactor to Promote Catalysis
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- 作者：Alasdair F. Bell ; Christopher F. Stratton ; Xujie Zhang ; Polina Novichenok ; Andrew A. Jaye ; Pravin A. Nair ; Sapan Parikh ; Richa Rawat ; Peter J. Tonge
- 刊名：Journal of the American Chemical Society
- 出版年：2007
- 出版时间：May 23, 2007
- 年：2007
- 卷：129
- 期：20
- 页码：6425 - 6431
- 全文大小：144K
- 年卷期：v.129,no.20(May 23, 2007)
- ISSN：1520-5126
文摘
InhA, the enoyl reductase from Mycobacterium tuberculosis, catalyzes the NADH-dependentreduction of trans-2-enoyl-ACPs. In the present work, Raman spectroscopy has been used to identifycatalytically relevant changes in the conformation of the nicotinamide ring that occur when NADH binds toInhA. For 4(S)-NADD, there is an 11 cm-1 decrease in the wavenumber of the C4-D stretching band(
fchars/nu.gif" BORDER=0 >C-D) and a 50% decrease in the width of this band upon binding to InhA. While a similar reduction in linewidth is observed for the corresponding band arising from 4(R)-NADD, fchars/nu.gif" BORDER=0 >C-D for this isomer increases 34cm-1 upon binding to InhA. These changes in fchars/nu.gif" BORDER=0 >C-D indicate that the nicotinamide ring adopts a boundconformation in which the 4(S)C-D bond is in a pseudoaxial orientation. Mutagenesis of F149, a conservedactive site residue close to the cofactor, demonstrates that this enzyme-induced modulation in cofactorstructure is directly linked to catalysis. In contrast to the wild-type enzyme, Raman spectra of NADD boundto F149A InhA resemble those of NADD in solution. Consequently, F149A is no longer able to optimallyposition the cofactor for hydride transfer, which correlates with the 30-fold decrease in kcat and 2-fold increasein D(V/KNADH) caused by this mutation. These studies thus substantiate the proposal that hydride transferis promoted by pseudoaxial positioning of the NADH pro-4S bond, and indicate that catalysis of substratereduction by InhA results, in part, from correct orientation of the cofactor in the ground state.
fchars/nu.gif" BORDER=0 >C-D) and a 50% decrease in the width of this band upon binding to InhA. While a similar reduction in linewidth is observed for the corresponding band arising from 4(R)-NADD, fchars/nu.gif" BORDER=0 >C-D for this isomer increases 34cm-1 upon binding to InhA. These changes in fchars/nu.gif" BORDER=0 >C-D indicate that the nicotinamide ring adopts a boundconformation in which the 4(S)C-D bond is in a pseudoaxial orientation. Mutagenesis of F149, a conservedactive site residue close to the cofactor, demonstrates that this enzyme-induced modulation in cofactorstructure is directly linked to catalysis. In contrast to the wild-type enzyme, Raman spectra of NADD boundto F149A InhA resemble those of NADD in solution. Consequently, F149A is no longer able to optimallyposition the cofactor for hydride transfer, which correlates with the 30-fold decrease in kcat and 2-fold increasein D(V/KNADH) caused by this mutation. These studies thus substantiate the proposal that hydride transferis promoted by pseudoaxial positioning of the NADH pro-4S bond, and indicate that catalysis of substratereduction by InhA results, in part, from correct orientation of the cofactor in the ground state.