Kandel M, Gornall A G, Lam L K, Kandel S I
Can J Biochem. 1975 May;53(5):599-608. doi: 10.1139/o75-081.
Partial inactivation of tau-dinitrophenylhistidine-200 human carbonic anhydrase B, induced by visible light, followed first order kinetics (k(app) = 6.05 times 10-2 min-1). After 50 min the tau-dinitrophenylhistidine (tau-DNP-histidine) content decreased to a negligible level, but the illuminated enzyme retained, at pH 7.6, approximately 9.2 percent of the esterase activity of the native enzyme. The following lines of evidence suggest that the loss of activity results from the destruction of tau-DNP-histidine-200. (1) No significant loss of amino acid other than tau-DNP-histidine was detected after illumination. (2) The rate of loss of activity correlated well with the loss of tau-DNP-histidine. (3) In the photooxidized enzyme the DNP moiety was retained but had lost the characteristic sensitivity of tau-DNP-histidine to nucleophilic attack. Titration of the illuminated enzyme with acetazolamide indicated that the residual activity is an intrinsic property of the modified enzyme. The chromatographically purified photooxidized enzyme migrated as a single band on isoelectrofocusing in polyacylamide gel, and at pH 7.6 possessed 7.5 percent esterase activity relative to the native enzyme. By establishing effective destruction of histidine-200, it can be concluded that neither the pi N nor, as previously shown, the tau N of histidine-200 is critical for the catalysis.
可见光诱导的tau - 二硝基苯基组氨酸 - 200人碳酸酐酶B的部分失活遵循一级动力学（表观速率常数k(app) = 6.05×10⁻² min⁻¹）。50分钟后，tau - 二硝基苯基组氨酸（tau - DNP - 组氨酸）含量降至可忽略不计的水平，但在pH 7.6时，光照后的酶保留了天然酶约9.2%的酯酶活性。以下证据表明活性丧失是由tau - DNP - 组氨酸 - 200的破坏引起的。（1）光照后未检测到除tau - DNP - 组氨酸外的其他氨基酸有显著损失。（2）活性丧失速率与tau - DNP - 组氨酸的损失密切相关。（3）在光氧化酶中，DNP部分保留，但失去了tau - DNP - 组氨酸对亲核攻击的特征敏感性。用乙酰唑胺滴定光照后的酶表明，残余活性是修饰酶的固有特性。经色谱纯化的光氧化酶在聚丙烯酰胺凝胶等电聚焦时迁移为单一条带，在pH 7.6时相对于天然酶具有7.5%的酯酶活性。通过确定组氨酸 - 200的有效破坏，可以得出结论，组氨酸 - 200的π氮和如先前所示的τ氮对催化作用都不关键。
