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.
Fourier-Transform Infrared Spectroscopy (FTIR) is a rapid structural technique used for determining structural changes, enzyme-inhibitor interactions and enzyme activity. Although FTIR is more rapid than time-consuming detailed structural techniques, such as X-ray crystallography, and provides more structural information in terms of bond strengths and mobility than UV-visible spectroscopy, direct interpretation of FTIR data still remains difficult. Molecular modelling, molecular dynamics and quantum mechanical techniques have been used to further interpret FTIR data. Acylated and native chymotrypsin have been used as a model system, since chymotrypsin is probably the best structurally characterised enzyme and extensive m I R studies on acylated chymotrypsins have been undertaken. The data has been interpreted as indicating that most inhibitors adopt multiple conformations once covalently bound to the enzyme. We are undertaking X-ray crystallographic simulation and quantum chemical studies to directly link the observed FTIR frequencies with structure. Molecular modelling and quantum chemical techniques, including Gaussian, Amber and MOPAC, have been utilised to produce models of the acylating agents N-trans-cinnamate and trans-0-hydroxymethylcinnamate, whilst molecular dynamics techniques are being used to provide models of the potential multiple conformations of these inhibitors bound to chymotrypsin. Crystallographic techniques are being used to validate these models. Small crystals have been grown of a-chymotrypsin with and without the presence of the inhibitor N-trans-cinnamoyl-imidazole (NTCI). Predictions of the FTIR frequencies for specific bonds in the inhibitor are being made using both semiempirical and ab initio quantum chemical techniques and comparisons with the experimental results are being undertaken.
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