Goodall Jonathan J, Booth Victoria K, Ashcroft Alison E, Wharton Christopher W
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Chembiochem. 2002 Jan 4;3(1):68-75. doi: 10.1002/1439-7633(20020104)3:1<68::AID-CBIC68>3.0.CO;2-D.
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.
用异吲哚酮酸酐对α-胰凝乳蛋白酶进行酰化反应生成的酰基酶已通过红外光谱进行了表征。在pH 7条件下进行酰化反应生成2-氨基苯甲酰基酶的速度很快（k = 5.57×10⁻² s⁻¹），而脱酰反应则慢得多（k = 3.7×10⁻⁵ s⁻¹）。已合成了[¹³C=O]标记的异吲哚酮酸酐形式，以构建[¹³C=O]减去[¹²C=O]的差示光谱；这些光谱突出了配体的羰基吸收，并消除了由蛋白质扰动引起的光谱效应。酰基酶的酯羰基带在波数1695 cm⁻¹处吸收，经去卷积分析表明它代表一种单一的、明确的构象。对一系列溶剂中2-氨基苯甲酸乙酯的模型研究表明，其羰基处于类似己烷的环境中（即非常非极性）。有人提出，羰基吸收的低波数是由于2-氨基与酯羰基氧之间存在分子内氢键；这导致酶中和非极性溶剂中羰基的极化。然而，鉴于脱酰反应缓慢，很明显酰基处于非生产性构象，与氧阴离子空穴没有相互作用，脱酰反应是从这种形式或一种次要的、不可见的形式发生的。红外数据得到了动力学电喷雾质谱测量的支持，该测量表明酰基酶是先前预期的那种，也得到了2-氨基苯甲酰基-α-胰凝乳蛋白酶的分子建模的支持。从pH依赖性测量得出的结论是，2-氨基的一般碱催化不参与脱酰反应。
