Time resolution of events in an enzyme's active site at 4 K and 300 K using resonance Raman spectroscopy

Abstract

ABSTRACT Resonance Raman spectra for enzyme-substrate intermediates of the type R(C=O)NHCH2C(S)S-Papain arereported in solution at 300 K and down to 4 K in ice matrices. Analysis reveals that the overall conformation of thesubstrate-enzyme bonds in the active site remains the same in the range 300 - 4 K but there are important minor spectralchanges. Some of these provide access to information on dynamical fluctuations occurring in the active site. Evidence forclosely lying fluctuating protein states driving fluctuations in the structure of the bound substrate is discussed. 2. INTRODUCTION Resonance Raman (RR) spectra for enzyme-substrate intermediates of the type R(C=O)NHCH2C(=S)S-Papainprovide vibrational data on those bonds in the active site undergoing catalytic transformation1 - namely those associatedwith the chromophoric -C(=S)S- group. Detailed structural and mechanistic information has been obtained from the RRdata and recent attention has focussed on extending the analysis to provide dynamical information. The approach has beento obtain RR spectra for the intermediates from 300 (in solution) to 4 K (in ice matrices) to characterize the effect on thevibrational spectrum of removing thermal energy from the protein matrix. In turn, this provides insight into the role ofthermal energy in driving the catalytic reaction.