Vibrations of the scissile C—O bond in an acyl-chymotrypsin observed by resonance Raman spectroscopy

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Abstract
5-Methylthienylacryloyl chymotrypsin has been prepared with 13C at the carbonyl and, separately, at the α carbon atoms of the acyl moiety. The isotopically labelled species confirm the assignments of several of the bands in the resonance Raman (RR) spectrum of the acyl-enzyme and show that the feature occurring near 1260 cm−1 involves the motion of the carbonyl carbon atom. It is likely that the 1260 cm−1 band is associated with the stretching vibration of the C—O bond in the =C—C(=O)—O— chymotrypsin moiety, i.e. the vibration of the bond being cleaved in the active site. A moderately intense 1260 cm−1 feature occurs only in the RR spectrum of the native acyl-enzyme and possible reasons are given for its appearance. The labelled species also confirm that the bands appearing near 1715 cm−1 in the RR spectrum of the acyl-enzyme are associated with the stretching mode of the acyl carbonyl, νC=O. This frequency is that expected for an unperturbed α, β-unsaturated ester. Additionally, RR data are presented for 5-methylthienylacrylic acid in its —COOH and —COO− forms labelled with 13C at the C=O and α carbon positions.