Conformational activation of acylpapains and acylcathepsin B's: resonance Raman and kinetic evidence.

Abstract

It is shown that, by extending the size of the substrate to meet papain's specificity requirement, conformational changes occur in bonds adjacent to the scissile linkage in the acyl enzyme. The resonance Raman (RR) spectra of the dithioacylpapains CH3OC(=O)-Phe-NHCH2C(=S)S-papain, CH3O(=O))-Gly-Phe-NHCH2C(=S)S-papain, and CH3OC(=O)-Gly-Gly-Phe-NHCH2C(=S)S-papain, formed from thiono ester substrates, are found to be very similar, indicating that each dithioacyl enzyme takes up an identical B-type conformation in the -NHCH2C(=S)SCH2-linkages. Moreover, the kcat and kcat/Km parameters for the reactions involving these intermediates are similar (in the ranges 0.51-0.67 s-1 and 44 100-68 200 M-1 s-1, respectively). For these reactions kcat = k3, the rate constant for deacylation. The strong structural similarities in the region undergoing catalytic transformation, taken with the similar k3's, show that placing glycine residues in papain's S3 and S4 subsites has little effect on the deacylation process. However, there are marked RR spectral and kinetic differences between the intermediates formed from these three substrates, all of which meet papain's requirement for Phe at the S2 subsite, and N-(beta-phenylpropionyl)glycine dithioacylpapain, C6H5(CH2)2C(=O)NHCH2C(=S)S-papain. The RR spectral differences indicate that changes occur in the torsional angles of the -NH-CH2-C(=S)-S-CH2- fragment upon going to the di-, tri-, and tetrapeptide-based substrates. The changes in these torsional angles are consistent with slight conformational activation since higher k3 values are found for the di-, tri-, and tetrapeptide-based substrates (0.51-0.67 s-1) compared to the k3 for C6H5(CH2)2C(=O)NHCH2C-(=S)S-papain of 0.165 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)