FINE-TUNED CONFORMATION OF DITHIOACYLPAPAIN INTERMEDIATES - INSIGHTS FROM RESONANCE RAMAN-SPECTROSCOPY

Interpretation of the resonance Raman (RR) spectrum of functioning N-b enzoylglycine dithioacylpapain, (C6H5C(=O)NHCH2C(=S)SCH2-papain) provi des an estimate of four torsion angles near the point of catalytic att ack. The torsion angles CN-CC, NC-CS(thiol), CS-CC, and SC-C25alpha(N) are-105, +15-degrees, gauche-, and P(H) (the hydrogen linked to C25al pha is in the transposition with respect to the thiol sulfur atom), re spectively. Spectral interpretation relies on spectra-structure correl ations derived from crystal structures of six N-acylglycine ethyl dith ioesters, viz. N-acetylglycine, N-(beta-phenylpropionyl)glycine, N-ben zoylglycine (two forms), N-(p-methylbenzoyl)glycine, and N-(p-chlorobe nzoyl)glycine ethyl dithioester, which have been examined by a combina tion of RR spectroscopic and X-ray crystallographic analyses. The stru ctural conclusions are reached using three marker bands, near 1130, 68 0, and 550 cm-1, in the RR spectrum of the dithioacylpapain and are su pported by the intensity pattern of three additional marker bands in t he 850-1050 cm-1 region. The value of +15-degrees derived for the NC-C S(thiol) torsion angle in the acyl enzyme is outside the range of -22. 2 to +9.5-degrees observed in model N-acylglycine ethyl dithioesters a nd lies in the direction of the NC-CS(thiol) torsion angle for the tra nsition state for deacylation. Thus, enzyme-substrate interactions cau se a modest distortion of at least one torsion angle in the direction of the reaction pathway. In addition, crystal structure data for the s ix N-acylglycine ethyl dithioesters are discussed, including the newly determined structure of N-(p-methylbenzoyl)glycine ethyl dithioester. All dithioesters adopt so-called B-type conformations with small valu es for the NC-CS(thiol) torsion angle and short N...S(thiol)nonbonded distances. The data reveal that correlated structural changes occur wi thin the common-C(=O)NHCH2C(=S)SCH2CH3 skeleton. These correlations in volve bond lengths (e.g., NC-CSS), bond angles (e.g., N-C-C), and tors ion angles (e.g., NC-CS(thiol)) and are a consequence of the N...S(thi ol) nonbonded interaction. Importantly, they provide a basis for predi cting the correlated changes in structural parameters in the dithioest er moiety in the active site of the enzyme as it undergoes transformat ion along the reaction pathway.