Markedly Different Acyl Papain Structures Deacylate at Similar Rates: Resonance Raman Spectroscopic and Kinetic Evidence

Article abstract of DOI:10.1021/ja00011a037

Resonance Raman (RR) spectroscopy has been used to determine the structure of the acyl group in a series of dithioacyl papains in which the side chain of the substrates P₁ amino acid residue has been extended from 2 (CH₃CH₂-) to 3 (CH₃CH₂CH₂-), and to 4 (CH₃CH₂CH₂CH₂-) carbon atoms in a linear chain. A conformational analysis was carried out on the corresponding ethyl ester model compounds, N-(methyloxycarbonyl)-L-phenylalanyl-L-ethylglycine, N-(methyloxycarbonyl)-L-phenylalanyl-L-norvaline, and N-(methyloxycarbonyl)-L-phenylalanyl-L-norleucine ethyl dithio esters, based on the RR spectra and known conformational states of glycine and alanine-based dithio esters. Comparison of the RR spectra of the model compounds with those of the corresponding N-(methyloxycarbonyl)-L-phenylalanyl-L-ethylglycine, -L-norvaline, and -L-norleucine dithioacyl papains shows that the acyl fragments adopt an A-like structure in the active site. An A-like structure is characterized by a large (near ±160°) nitrogen to thiol sulfur torsional angle about the NHCHR'-CS single bond. This conformation is in marked contrast to that found for N-acylglycine dithioacyl papains which have a small (near ±15°) NHCH₂-CS(thiol) torsional angle in the P₁ residue giving rise to the so-called B conformer. Thus we have evidence that the two classes of substrate give rise to two substantially different acyl group structures in the active site. However, for the ethylglycine, norvaline, and norleucine dithioacyl papains the deacylation rate constants (k_cat's) are only ca. 3 times greater than k_cat for the most reactive W-acylglycine substrate. Thus deacylation can occur from both A- and B-type dithioacyl papains with only a small kinetic penalty for the latter. The existence of an A-type conformer in the active site and the need to maintain binding in the oxyanion hole raise the possibility that the acyl group is binding backwards, i.e. in the S₁' and S₂' binding sites.