Electric fields in active sites: Substrate switching from null to strong fields in thiol- and selenol-subtilisins

Although known to be important factors in promoting catalysis, electric fie ld effects in enzyme active sites are difficult to characterize from an exp erimental standpoint. Among optical probes of electric fields, Raman spectr oscopy has the advantage of being able to distinguish electronic ground-sta te and excited-state effects. Earlier Raman studies on acyl derivatives of cysteine proteases [Doran, J, D., and Carey, P. R. (1996) Biochemistry 35, 12495-502], where the acyl group has extensive pi-electron conjugation, sho wed that electric field effects in the active site manifest themselves by p olarizing the pi-electrons of the acyl group. Polarization gives rise to la rge shifts in certain Raman bands, e.g., the C=C stretching band of the alp ha,beta-unsaturated acyl group, and a large red shift in the absorption max imum. It was postulated that a major source of polarization is the alpha-he lix dipole that originates from the alpha-helix terminating at the active-s ite cysteine of the cysteine protease family. In contrast, using the acyl g roup 5-methylthiophene acryloyl (5-MTA) as an active-site Raman probe, acyl enzymes of thiol- or selenol-subtilisin exhibit no polarization even thoug h the acylating amino acid is at the terminus of an alpha-helix. Quantum me chanical calculations on 5-MTA ethyl thiol and selenol ethyl esters allowed us to identify the conformational states of these molecules along with the ir corresponding vibrational signatures. The Raman spectra of 5-MTA thiol a nd selenol subtilisins both showed that the acyl group binds in a single co nformation in the active site that is s-trans about the =C-C=O single bond. Moreover, the positions of the C=C stretching bands show that the acyl gro up is not experiencing polarization. However, the release of steric constra ints in the active site by mutagenesis, by creating the N155G form of selen ol-subtilisin and the P225A form of thiol-subtilisin, results in the appear ance of a second conformer in the active Sites that is s-cis about the =C-C =O bond. The Raman signature of this second conformer indicates that it is strongly polarized with a permanent dipole being set up through the acyl gr oup's pi-electron chain. Molecular modeling for 5-MTA in the active sites o f selenol-subtilisin and N155G selenol-subtilisin confirms the findings fro m Raman spectroscopic studies and identifies the active-site features that give rise to polarization. The determinants of polarization appear to be st rong electron pull at the acyl carbonyl group by a combination of hydrogen bonds and the field at the N-terminus of the alpha-helix and electron push from a negatively charged group placed at the opposite end of the chromopho re.