Khalifah R G, Edsall J T
Proc Natl Acad Sci U S A. 1972 Jan;69(1):172-6. doi: 10.1073/pnas.69.1.172.
A stop-flow kinetic study was performed on the carbon dioxide hydration activity of the human carbonic anhydrase B isoenzyme carboxymethylated at its histidine(200), and of the human C isoenzyme carboxyketoethylated at its histidine(63). The Michaelis-Menten parameters determined between pH 5.6 and 8.7 showed striking differences between the native and the alkylated enzymes, as well as between the modified enzymes themselves. The alkylations caused: (i) a decrease in the k(cat) values, particularly marked for the carboxymethylated B isoenzyme, (ii) a change in the apparent pK of the k(cat) curves, and (iii) a dependence of K(m) on pH, for the alkylated enzymes, in contrast to the pH-independent K(m) values of the native enzymes. The CO(2) hydration and esterase activities of the carboxymethyl B isoenzyme differ markedly in their pH dependence. A kinetic mechanism, which is found to be compatible with all the present observations, is proposed. The results indicate that the modifiable histidine residues do not play an essential role in the catalytic mechanism of the native carbonic anhydrases, but they may well influence the enzyme activity in a secondary role.
对在其组氨酸（200）处羧甲基化的人碳酸酐酶B同工酶以及在其组氨酸（63）处羧基酮乙基化的人C同工酶的二氧化碳水合活性进行了停流动力学研究。在pH 5.6至8.7之间测定的米氏参数显示，天然酶与烷基化酶之间以及修饰酶本身之间存在显著差异。烷基化导致：（i）k(cat)值降低，尤其是羧甲基化的B同工酶，（ii）k(cat)曲线的表观pK发生变化，以及（iii）对于烷基化酶，K(m)对pH有依赖性，这与天然酶的pH无关的K(m)值形成对比。羧甲基化B同工酶的CO₂水合和酯酶活性在pH依赖性方面有显著差异。提出了一种与所有当前观察结果相符的动力学机制。结果表明，可修饰的组氨酸残基在天然碳酸酐酶的催化机制中不发挥关键作用，但它们很可能以次要作用影响酶活性。
