Jolley M E, Gray C J
Carbohydr Res. 1976 Jul;49:361-70. doi: 10.1016/s0008-6215(00)83153-5.
The pH-dependence of the photo-oxidation of L-tryptophan, in the presence of Rose Bengal and Methylene Blue, has been investigated. True, initial rate constants were determined in order to circumvent errors due to secondary processes. Photo-oxidation of glycoamylase I from A. niger in the presence of Methylene Blue or Rose Bengal resulted in a pH-dependent loss of enzymic activity, which was analogous to the destruction of free L-tryptophan during photo-oxidation. The loss of enzymic activity was closely associated with the destruction of tryptophan residues in the enzyme. Significant protection of both enzymic activity and tryptophanyl residues in the enzyme molecule was achieved by performing the photo-oxidation in the presence of maltose, which is a substrate for the enzyme. The tryptophanyl residues of glucoamylase I, which had been inactivated by reaction of its carboxylic acid residues with glycine methyl ester in the presence of a water-soluble carbodi-imide, were also substantially protected by maltose. It is concluded that the active centre of glucoamylase I is a cleft lined with tryptophanyl residues that participate in the binding of the substrate. One or more carboxylic acid residues are involved in bond cleavage.
研究了在孟加拉玫瑰红和亚甲蓝存在下，L-色氨酸光氧化反应的pH依赖性。确实，为了规避次级过程导致的误差，测定了真实的初始速率常数。在亚甲蓝或孟加拉玫瑰红存在下，黑曲霉糖化淀粉酶I的光氧化导致酶活性出现pH依赖性损失，这与光氧化过程中游离L-色氨酸的破坏类似。酶活性的损失与酶中色氨酸残基的破坏密切相关。通过在麦芽糖（该酶的一种底物）存在下进行光氧化，酶分子中的酶活性和色氨酸残基均得到了显著保护。糖化淀粉酶I的色氨酸残基，在水溶性碳二亚胺存在下，其羧酸残基与甘氨酸甲酯反应而失活，也受到了麦芽糖的显著保护。得出的结论是，糖化淀粉酶I的活性中心是一个由参与底物结合的色氨酸残基排列而成的裂隙。一个或多个羧酸残基参与键的断裂。
