Electric Fields in Active Sites: Substrate Switching from Null to Strong Fields in Thiol- and Selenol-Subtilisins
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- 作者：Deendayal Dinakarpandian ; Bhami C. Shenoy ; Donald Hilvert ; Duncan E. McRee ; Michele McTigue ; and Paul R. Carey
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：May 18, 1999
- 年：1999
- 卷：38
- 期：20
- 页码：6659 - 6667
- 全文大小：105K
- 年卷期：v.38,no.20(May 18, 1999)
- ISSN：1520-4995
文摘
Although known to be important factors in promoting catalysis, electric field effects in enzymeactive sites are difficult to characterize from an experimental standpoint. Among optical probes of electricfields, Raman spectroscopy has the advantage of being able to distinguish electronic ground-state andexcited-state effects. Earlier Raman studies on acyl derivatives of cysteine proteases [Doran, J. D., andCarey, P. R. (1996) Biochemistry 35, 12495-502], where the acyl group has extensive 
-electronconjugation, showed that electric field effects in the active site manifest themselves by polarizing the-electrons of the acyl group. Polarization gives rise to large shifts in certain Raman bands, e.g., theC=C stretching band of the 
,
-unsaturated acyl group, and a large red shift in the absorption maximum.It was postulated that a major source of polarization is the -helix dipole that originates from the -helixterminating at the active-site cysteine of the cysteine protease family. In contrast, using the acyl group5-methylthiophene acryloyl (5-MTA) as an active-site Raman probe, acyl enzymes of thiol- or selenol-subtilisin exhibit no polarization even though the acylating amino acid is at the terminus of an -helix.Quantum mechanical calculations on 5-MTA ethyl thiol and selenol ethyl esters allowed us to identifythe conformational states of these molecules along with their corresponding vibrational signatures. TheRaman spectra of 5-MTA thiol and selenol subtilisins both showed that the acyl group binds in a singleconformation in the active site that is s-trans about the =C-C=O single bond. Moreover, the positionsof the C=C stretching bands show that the acyl group is not experiencing polarization. However, therelease of steric constraints in the active site by mutagenesis, by creating the N155G form of selenol-subtilisin and the P225A form of thiol-subtilisin, results in the appearance of a second conformer in theactive sites that is s-cis about the =C-C=O bond. The Raman signature of this second conformer indicatesthat it is strongly polarized with a permanent dipole being set up through the acyl group's -electronchain. Molecular modeling for 5-MTA in the active sites of selenol-subtilisin and N155G selenol-subtilisinconfirms the findings from Raman spectroscopic studies and identifies the active-site features that giverise to polarization. The determinants of polarization appear to be strong electron pull at the acyl carbonylgroup by a combination of hydrogen bonds and the field at the N-terminus of the -helix and electronpush from a negatively charged group placed at the opposite end of the chromophore.
-electronconjugation, showed that electric field effects in the active site manifest themselves by polarizing the-electrons of the acyl group. Polarization gives rise to large shifts in certain Raman bands, e.g., theC=C stretching band of the ,-unsaturated acyl group, and a large red shift in the absorption maximum.It was postulated that a major source of polarization is the -helix dipole that originates from the -helixterminating at the active-site cysteine of the cysteine protease family. In contrast, using the acyl group5-methylthiophene acryloyl (5-MTA) as an active-site Raman probe, acyl enzymes of thiol- or selenol-subtilisin exhibit no polarization even though the acylating amino acid is at the terminus of an -helix.Quantum mechanical calculations on 5-MTA ethyl thiol and selenol ethyl esters allowed us to identifythe conformational states of these molecules along with their corresponding vibrational signatures. TheRaman spectra of 5-MTA thiol and selenol subtilisins both showed that the acyl group binds in a singleconformation in the active site that is s-trans about the =C-C=O single bond. Moreover, the positionsof the C=C stretching bands show that the acyl group is not experiencing polarization. However, therelease of steric constraints in the active site by mutagenesis, by creating the N155G form of selenol-subtilisin and the P225A form of thiol-subtilisin, results in the appearance of a second conformer in theactive sites that is s-cis about the =C-C=O bond. The Raman signature of this second conformer indicatesthat it is strongly polarized with a permanent dipole being set up through the acyl group's -electronchain. Molecular modeling for 5-MTA in the active sites of selenol-subtilisin and N155G selenol-subtilisinconfirms the findings from Raman spectroscopic studies and identifies the active-site features that giverise to polarization. The determinants of polarization appear to be strong electron pull at the acyl carbonylgroup by a combination of hydrogen bonds and the field at the N-terminus of the -helix and electronpush from a negatively charged group placed at the opposite end of the chromophore.