Elbaum D, Nagel R L
J Biol Chem. 1981 Mar 10;256(5):2280-3.
The hydrolysis of p-nitrophenyl acetate (p-NPA) is catalyzed by many proteins. We have observed that oxyhemoglobin A also exhibits esterase activity with a rate intermediate between that of bovine albumin and carbonic anhydrase. Kinetic studies of this reaction revealed that the rate of hydrolysis of p-NPA in the presence of oxy Hb S was approximately 2 times slower than that of oxy Hb A. There is general agreement that the catalytic effect of peptides and proteins on the hydrolysis of p-NPA is mediated by histidines. Oxy Hb Deer Lodge (His beta 2 leads to Arg) hydrolyzes p-NPA at a rate approaching that of oxy Hb S. Oxy Hb F, in turn, exhibits a rate indistinguishable from that of oxy Hb A. The effect of 2,3-diphosphoglyceric acid on the reaction is consistent with the participation of His beta 2 in this catalytic effect. The pH dependence of the reaction and studies with free amino acids also lend support to the involvement of a histidine residue. These results point to subtle conformational differences between oxy Hb S and oxy Hb A in solution, probably involving His beta 2 and/or its microenvironment. The catalytic hydrolysis of p-NPA can be considered a useful probe of conformational states of macromolecules.
对硝基苯乙酸酯（p-NPA）的水解可由多种蛋白质催化。我们观察到，氧合血红蛋白A也表现出酯酶活性，其速率介于牛血清白蛋白和碳酸酐酶之间。对该反应的动力学研究表明，在氧合Hb S存在下，p-NPA的水解速率比氧合Hb A慢约2倍。人们普遍认为，肽和蛋白质对p-NPA水解的催化作用是由组氨酸介导的。氧合鹿屋血红蛋白（Hisβ2突变为Arg）水解p-NPA的速率接近氧合Hb S。相反，氧合Hb F的水解速率与氧合Hb A无法区分。2,3-二磷酸甘油酸对该反应的影响与Hisβ2参与这种催化作用一致。反应的pH依赖性以及对游离氨基酸的研究也支持了组氨酸残基的参与。这些结果表明，溶液中的氧合Hb S和氧合Hb A之间存在细微的构象差异，可能涉及Hisβ2和/或其微环境。p-NPA的催化水解可被视为一种研究大分子构象状态的有用探针。
