EFFECT OF SPECIFICITY ON LIGAND CONFORMATION IN ACYL-CHYMOTRYPSINS

I.r. difference spectroscopy combined with C-13 and O-18 double-isotop e substitution was used to examine the ester acyl carbonyl stretching vibration of hydrocinnamoyl-chymotrypsin. A single acyl carbonyl stret ching band was observed at 1731 cm(-1). This contrasts with previous i .r. and resonance Raman spectroscopic studies of a number of trans-3-a rylacryloyl-chymotrypsins which showed two acyl carbonyl stretching ba nds in the region of 1700 cm(-1), which were proposed to represent pro ductive and non-productive conformations of the acyl-enzyme. The singl e acyl carbonyl band for hydrocinnamoyl-chymotrypsin suggests only a s ingle 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 polariz ation and thereby catalytic-rate acceleration. Thus, in view of the ap parent lack of such hydrogen-bonding in hydrocinnamoyl-chymotrypsin, i t 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 pop ulation of lower stretching frequency for hydrocinnamoyl-chymotrypsin, but that this is too small to be discerned because of either a relati vely high deacylation rate or an unfavourable conformational equilibri um. The single acyl carbonyl band for hydrocinnamoyl-chymotrypsin is s ignificantly broader than those for trans-3-arylacryloyl-chymotrypsins , indicating that this group is more conformationally mobile and dispe rsed in the former. This can be correlated with the absence of acyl ca rbonyl hydrogen-bonding in hydrocinnamoyl-chymotrypsin, and with the m uch greater flexibility of the saturated hydrocinnamoyl group than uns aturated trans-3-arylacryloyl. This flexibility is presumably the reas on why hydrocinnamoyl-chymotrypsin is more specific than trans-3-aryla cryloyl-chymotrypsins. Resonance Raman spectroscopy is limited to the non-specific trans-3-arylacryloyl-chymotrypsins because of its chromop horic requirement, whereas i.r. may be used to examine non-chromophori c more specific acyl-enzymes such as hydrocinnamoyl chymotrypsin. The results presented in this paper suggest that trans-3-arylacryloyl-chym otrypsins are atypical.