Smith R, Brereton I M, Chai R Y, Kent S B
Department of Biochemistry, University of Queensland, Australia.
Nat Struct Biol. 1996 Nov;3(11):946-50. doi: 10.1038/nsb1196-946.
Chemical synthesis was used to prepare the HIV-1 protease specifically 13C-labelled in the catalytically essential Asp 25 in each monomer. The NMR chemical shift of the 13C-enriched homodimeric enzyme was measured in the presence of the inhibitor pepstatin, a mimic of the tetrahedral intermediate formed in enzyme catalysis. In this complex, the catalytic carboxyls do not titrate in the pH range where the enzyme is active; throughout the range pH 2.5-6.5, one Asp 25 side chain is protonated and the other deprotonated. By contrast, in the absence of inhibitor the two Asp side chains are chemically equivalent and both deprotonated at pH6, the optimum for enzymatic activity. These direct observations of the chemical properties of the catalytic apparatus of the enzyme provide concrete information on which to base the design of improved HIV-1 protease inhibitors.
利用化学合成法制备了在每个单体中催化必需的天冬氨酸25处特异性13C标记的HIV-1蛋白酶。在抑制剂胃蛋白酶抑素（一种酶催化过程中形成的四面体中间体模拟物）存在的情况下，测量了13C富集的同二聚体酶的核磁共振化学位移。在这种复合物中，催化羧基在酶活性的pH范围内不发生滴定；在pH 2.5 - 6.5范围内，一个天冬氨酸25侧链质子化，另一个去质子化。相比之下，在没有抑制剂的情况下，两个天冬氨酸侧链在化学上是等效的，并且在pH6（酶活性的最佳pH）时都去质子化。这些对酶催化装置化学性质的直接观察为设计改进的HIV-1蛋白酶抑制剂提供了具体信息。
