CHARACTERIZATION OF TRANS-5-METHYLTHIENYLACRYLOYL AND CIS-5-METHYLTHIENYLACRYLOYL CHYMOTRYPSIN USING RAMAN DIFFERENCE SPECTROSCOPY, NMR, AND KINETICS - CARBONYL ENVIRONMENT AND REACTIVITY

The interpretation of the resonance Raman (RR) data for chromophoric a cyl serine proteases is rendered complex due to the fact that the almo st-equal-to 350-nm laser beam used to generate the RR spectrum causes the substrate to photoisomerize in the active site. As a result, a ste ady-state population of dark (present before irradiation) and light (p hotoinduced) conformers is set up. In the present work, 2D NMR experim ents are used to confirm that the photoinduced conformer of 5-methylth ienylacryloyl chymotrypsin is cis about the acryloyl -C=C- linkage, wh ereas it is trans for the dark conformer. Using 488.0- or 514.5-nm exc ited Raman difference spectroscopy the normal Raman spectra, under non photolyzing conditions, of the pure cis and trans forms of the acyl ch ymotrypsins are reported for the first time. A number of characteristi c bands for the two forms are identified, e.g., the bands at 1616, 147 0, and 1534 cm-1 for the trans form of the intermediate occur at 1611, 1419, and 1527 cm-1 in the cis form. In particular nu(C=O) for the ac yl carbonyl occurs at 1727 and 1697 cm-1 for the cis and trans forms, respectively. The two forms have markedly different reactivities with the trans isomer deacylating 5000 times faster than the cis. These spe ctroscopic and kinetic properties contrast sharply with the behavior o f the model compound 5-methylthienylacryloyl methyl ester. The cis and trans forms of this ester in CCl4 have values for nu(C=O) at 1720 +/- 2 cm-1, and the intrinsic reactivities of the cis and trans methyl es ters to base catalyzed hydrolysis are very similar, with k(OH-) of 0.0 062 and 0.013 s-1 in 1.0 M NaOH, respectively. Thus, the differential reactivities and nu(C=O) observed for the cis and trans acyl chymotryp sins are caused by differential interactions with the enzyme's active site and are not due to the intrinsic properties of the cis and trans form of the acyl group.