Abstract

By creating a resonance Raman probe in the active site of an enzyme, it is possible to obtain the vibrational spectrum associated with those bonds undergoing catalytic transformation. The approach involves reacting thionoesters RC(= S)OCH₃ with a class of enzymes known as cysteine proteases which have an essential SH group in their active sights HS-enzyme. The reaction produces an intermediate RC(= S) S-enzyme which is a dithioester with a λ_max near 315 nm. The 324-nm excited RR spectra of the dithioester provide a wealth of detail on the substrate during catalysis; the confirmation of the substrate in the active sight can be monitored and characterized, structure rate constant relationships developed, reaction pathways mapped, and evidence sought for geometric distortion. The novel findings stemming from the RR data are difficult to reconcile with the conventional view of enzyme mechanism.

© 1986 Optical Society of America