Machuga E, Klapper M H
Biochim Biophys Acta. 1977 Apr 12;481(2):526-41. doi: 10.1016/0005-2744(77)90285-6.
The pH-dependence of RNAase A and of Ntau-carboxymethylhistidine-12-RNAase (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase) catalysis was studied. Apparent acid dissociation constants were obtained by least squares analysis of the kinetics data. These dissociation constants were compared with pKa values of model imidazole compounds, and with pKa values of histidine residues 12 and 119 on the protein. The shapes of the kcat versus pH profiles for RNAase A and its carboxymethyl derivative are very similar, from which it is concluded that the mechanism of catalysis is closely similar in the two proteins. Apparent pKa values obtained from the kinetic data are higher for the carboxymethylated protein than for RNAase A, as are the pKa values of residues 12 and 119. The similar shifts are consistent with the conclusions that both these residues are functionally significant in native and modified enzyme, and that an unblocked tau-nitrogen on histidine-12 is not essential for activity. From the enzyme's catalytic dependence on pH, and the NMR determined pKa values we propose that histidine 12 and 119 function catalytically in their basic and acidic forms respectively.
研究了核糖核酸酶A和Nτ-羧甲基组氨酸-12-核糖核酸酶（核糖核酸3'-嘧啶基寡核苷酸水解酶）催化作用的pH依赖性。通过对动力学数据进行最小二乘法分析获得表观酸解离常数。将这些解离常数与咪唑模型化合物的pKa值以及蛋白质上组氨酸残基12和119的pKa值进行比较。核糖核酸酶A及其羧甲基衍生物的kcat与pH曲线形状非常相似，由此得出结论，两种蛋白质的催化机制非常相似。从动力学数据获得的表观pKa值，羧甲基化蛋白质比核糖核酸酶A更高，残基12和119的pKa值也是如此。类似的变化与以下结论一致：这两个残基在天然酶和修饰酶中均具有功能重要性，并且组氨酸-12上未被阻断的τ-氮对于活性并非必不可少。根据酶对pH的催化依赖性以及核磁共振测定的pKa值，我们提出组氨酸12和119分别以其碱性和酸性形式发挥催化作用。
