Di Donato A, Cafaro V, Romeo I, D'Alessio G
Dipartimento di Chimica Organica e Biologica, Università di Napoli Federico II, Naples, Italy.
Protein Sci. 1995 Aug;4(8):1470-7. doi: 10.1002/pro.5560040804.
Residues P19, L28, C31, and C32 have been implicated (Di Donato A, Cafaro V, D'Alessio G, 1994, J Biol Chem 269:17394-17396; Mazzarella L, Vitagliano L, Zagari A, 1995, Proc Natl Acad Sci USA: forthcoming) with key roles in determining the dimeric structure and the N-terminal domain swapping of seminal RNase. In an attempt to have a clearer understanding of the structural and functional significance of these residues in seminal RNase, a series of mutants of pancreatic RNase A was constructed in which one or more of the four residues were introduced into RNase A. The RNase mutants were examined for: (1) the ability to form dimers; (2) the capacity to exchange their N-terminal domains; (3) resistance to selective cleavage by subtilisin; and (4) antitumor activity. The experiments demonstrated that: (1) the presence of intersubunit disulfides is both necessary and sufficient for engendering a stably dimeric RNase; (2) all four residues play a role in determining the exchange of N-terminal domains; (3) the exchange is the molecular basis for the RNase antitumor action; and (4) this exchange is not a prerequisite in an evolutionary mechanism for the generation of dimeric RNases.
残基P19、L28、C31和C32被认为（迪多纳托A、卡法罗V、达莱西奥G，1994年，《生物化学杂志》269:17394 - 17396；马扎雷拉L、维塔利阿诺L、扎加里A，1995年，《美国国家科学院院刊》：即将发表）在决定精浆核糖核酸酶的二聚体结构和N端结构域交换方面起关键作用。为了更清楚地了解这些残基在精浆核糖核酸酶中的结构和功能意义，构建了一系列胰核糖核酸酶A的突变体，其中将这四个残基中的一个或多个引入核糖核酸酶A中。对这些核糖核酸酶突变体进行了以下检测：（1）形成二聚体的能力；（2）交换其N端结构域的能力；（3）对枯草杆菌蛋白酶选择性切割的抗性；以及（4）抗肿瘤活性。实验表明：（1）亚基间二硫键的存在对于产生稳定的二聚体核糖核酸酶既是必要的也是充分的；（2）所有这四个残基在决定N端结构域的交换中都起作用；（3）这种交换是核糖核酸酶抗肿瘤作用的分子基础；以及（4）这种交换在二聚体核糖核酸酶产生的进化机制中不是先决条件。
