New Techniques for the Resonance Raman Spectroscopy of Enzyme-Substrate Intermediates

Two new techniques are described for the study of intermediates formed in enzyme-substrate complexes by resonance Raman spectroscopy (RRS). The first technique uses an ultraviolet line from a YAG laser which is obtained by frequency doubling to 266nm and then shifting by means of stimulated antiStokes Raman scattering to 239nm. The advantage of this line is that it falls in an absorption minimum for the aromatic residues, tyrosine and tryptophan, which permits the observation of resonance Raman spectrum of intermediates whose absorption maxima fall within the range 225-245nm. In order not to decompose the sample and yet conserve it a slow flow technique has been devised which avoids the use of a quartz window. The method is illustrated with the complex formed from a simple oligopeptide and the enzyme, papain. Excellent ultraviolet RR spectra are obtained from the enzyme, the substrate and the complex. By computer subtraction of the component spectra from the spectra of the complex, the presence of a new band at 1680 wavenumbers is seen which is ascribed to the carbonyl band of the acyl group. Most studies of enzyme-substrate complexes by RRS uses a procedure in which the components are mixed and flowed through the laser beam. This results in the use of large amounts of enzyme which is not always available. To overcome this difficulty we have devised a laser Raman microscope set up in which a microcrystal of the enzyme is mounted in a special cell under the microscope and the chromophoric substrate is flowed over the crystal resulting in the build-up of and intermediate whose spectrum can be taken. This is illustrated with crystals of chymotrypsin .