Effect of specificity on ligand conformation in acyl-chymotrypsins Available to Purchase

I.r. difference spectroscopy combined with 13C and 18O double-isotope substitution was used to examine the ester acyl carbonyl stretching vibration of hydrocinnamoyl-chymotrypsin. A single acyl carbonyl stretching band was observed at 1731 cm-1. This contrasts with previous i.r. and resonance Raman spectroscopic studies of a number of trans-3-arylacryloyl-chymotrypsins which showed two acyl carbonyl stretching bands in the region of 1700 cm-1, which were proposed to represent productive and non-productive conformations of the acyl-enzyme. The single acyl carbonyl band for hydrocinnamoyl-chymotrypsin suggests only a single conformation, and the comparatively high frequency of this band implies little or no hydrogen-bonding to this carbonyl group. Enzymic hydrogen-bonding to the acyl carbonyl is believed to give bond polarization and thereby catalytic-rate acceleration. Thus, in view of the apparent lack of such hydrogen-bonding in hydrocinnamoyl-chymotrypsin, it should be the case that this acyl-chymotrypsin is less specific than trans-3-arylacryloyl-chymotrypsins, whereas the opposite is true. It is therefore proposed that there may be a productive acyl carbonyl population of lower stretching frequency for hydrocinnamoyl-chymotrypsin, but that this is too small to be discerned because of either a relatively high deacylation rate or an unfavourable conformational equilibrium. The single acyl carbonyl band for hydrocinnamoyl-chymotrypsin is significantly broader than those for trans-3-arylacryloyl-chymotrypsins, indicating that this group is more conformationally mobile and dispersed in the former. This can be correlated with the absence of acyl carbonyl hydrogen-bonding in hydrocinnamoyl-chymotrypsin, and with the much greater flexibility of the saturated hydrocinnamoyl group than unsaturated trans-3-arylacryloyl. This flexibility is presumably the reason why hydrocinnamoyl-chymotrypsin is more specific than trans-3-arylacryloyl-chymotrypsins. Resonance Raman spectroscopy is limited to the non-specific trans-3-arylacryloyl-chymotrypsins because of its chromophoric requirement, whereas i.r. may be used to examine non-chromophoric more specific acyl-enzymes such as hydrocinnamoyl-chymotrypsin. The results presented in this paper suggest that trans-3-arylacryloyl-chymotrypsins are atypical.