Relaxed and perturbed substrate conformations in enzyme active sites: evidence from multichannel resonance raman spectra.

A diode array based multichannel Raman spectrometer has made it possible to record complete, high quality, resonance Raman (RR) spectra of enzyme-substrate intermediates. The intermediates are dithioacylpapains in which the acyl group is either N-benzoylglycine or N-(beta-phenylpropionyl)glycine. RR data are reported for the unlabeled dithioacylpapains as well as for the intermediates labeled separately with ND, 15N, and 13C = S in the glycine residue. Comparison of the results for the dithioacylpapains with that of the corresponding labeled glycine ethyl dithioesters [Lee, H., Storer, A. C., & Carey, P. R. (1983) Biochemistry (preceding paper in this issue)] leads to the conclusion that for both substrates in the active site the dihedral angles in the glycine NH-C-C(= S) linkages assume an essentially relaxed type B conformation. Similarly, there is no evidence for distortion about the C(= O)-NH peptide bond which links the P1 and P2 sites on the substrate. However, for the N-benzoylglycine case there is evidence for some conformational distortion in the -S-C-C cysteine linkages. The present data favor a single homogeneous conformational population about the substrates' NH-C-C(= S) bonds in the native dithioacylpapains. However, below pH 3.0 the dithioacyl enzymes denature and the RR spectra of the 13C = S substituted species confirm that the conformational population reverts to the mixture of conformers A and B found for the corresponding ethyl dithioesters in solution.