Hydrogen-bonding in 2-aminobenzoyl-alpha-chymotrypsin formed by acylation of the enzyme with isatoic anhydride: IR and mass spectroscopic studies.

The acyl-enzyme formed upon acylation of alpha-chymotrypsin with isatoic anhydride has been characterised by infrared spectroscopy. Acylation at pH 7 to yield the 2-aminobenzoyl-enzyme is rapid (k = 5.57x 10(-2)s(-1)), while deacylation is much slower (k =3.7 x 10(-5)10(-2) (s-). The [1C=O]-labelled form of isatoic anhydride has been synthesised, to allow construction of [72C=O]- minus [13C=O]difference spectra; these highlight the carbonyl absorbance of the ligand and eliminate spectral effects that arise from protein perturbation. The ester carbonyl band of the acyl-enzyme absorbs at a wavenumber of 1695cm(-1) and has been shown by deconvolution analysis to represent a single, well-defined conformation. Model studies of ethyl 2-aminobenzoate in a range of solvents show that its carbonyl group is in a hexane-like environment (that is, very nonpolar). It is proposed that the low wavenumber of the carbonyl absorbance arises from the presence of an internal hydrogen bond between the 2-amino group and the ester carbonyl oxygen; this leads to polarisation of the carbonyl group both in the enzyme and in nonpolar solvents. However, in view of the slow deacylation, it is clear that the acyl group is in a nonproductive conformation, with no interaction with the oxyanion hole, and that deacylation occurs from this form or from a minor, invisible form. The infrared data have been supported by kinetic electrospray mass spectroscopic measurements, which demonstrate that the acyl-enzyme is that previously anticipated, and by molecular modelling of 2-aminobenzoyl-alpha-chymotrypsin. It is concluded from pH-dependence measurements that general base catalysis by the 2-amino group is not involved in deacylation.