The structure and dynamics of enzyme-substrate reaction intermediates determined by resonance Raman spectroscopy
The two principal means of obtaining the resonance Raman (RR) spectra of enzyme-substrate complexes are discussed. The first employs substrates which contain extended ¶-electron systems, such as cinnamic or furylacrylic acid. The spectral data for these substrates bound to serine or cysteine proteinases can be interpreted in terms of a model in which the delocalised it-electrons become polarised. The second method of employing the RR technique creates a dithioester chromophore at the point where the substrate is covalently linked, for a short time, to the enzyme. By this means it is possible to obtain the vibrational spectrum associated with those bonds undergoing catalytic transformation. The RR spectra provide detailed information on the conformation of the enzyme-bound substrate and allow us to discuss conformer selection and substrate distortion and strain. Combined X-ray crystallographic and Raman studies on suitable model compounds are important in setting up the spectra-structure correlations needed for spectral interpretation. These studies also introduce a dynamical concept by suggesting how structural changes occur along the reaction pathway. Complementary dynamical information is elicited from combined kinetic and RR data; the data from these two techniques provide a very detailed structure-kinetic correlation and show how the breaking of a single atom-atom contact in the active site is associated with an individual rate constant for the reaction. Papain is the archetype enzyme used for the dithioester work, but the approach has been extended to other plant cysteine proteinases and to the mammalian enzyme cathepsin B.