Tanizawa K, Nakano M, Kanaoka Y
Biochim Biophys Acta. 1987 Jul 7;913(3):292-9. doi: 10.1016/0167-4838(87)90138-5.
The preparation of fluorescence labeled acyl enzymes (Streptomyces griseus trypsin) was successfully carried out using specific trypsin substrates, 'inverse substrates'. The topographical analysis of the structures of the area around the active site was carried out by measuring the fluorescence spectra of the acyl enzyme preparations and these results were compared with those of bovine trypsin. It was found that the polarity of the active site vicinity at pH 5 was similar to that of bovine trypsin, whereas considerable differences were noticed at lower pH as a result of pH-induced transformation. Conformational changes of the active site induced by the interaction with the specific ligand were analyzed from the fluorescence spectra. In these responses the two enzymes were quite distinguishable. The two enzymes active sites were also different in the energy transfer experiments. The spatial arrangements of the catalytic residues relative to the intrinsic tryptophan residues were suggested to be substantially different for the two enzymes.
使用特定的胰蛋白酶底物“反向底物”成功制备了荧光标记的酰基酶（灰色链霉菌胰蛋白酶）。通过测量酰基酶制剂的荧光光谱对活性位点周围区域的结构进行了拓扑分析，并将这些结果与牛胰蛋白酶的结果进行了比较。结果发现，pH 5时活性位点附近的极性与牛胰蛋白酶相似，而在较低pH值下，由于pH诱导的转变，出现了显著差异。从荧光光谱分析了与特定配体相互作用诱导的活性位点构象变化。在这些反应中，两种酶非常容易区分。在能量转移实验中，两种酶的活性位点也不同。对于这两种酶，催化残基相对于内在色氨酸残基的空间排列被认为有很大差异。
