Panov K I, Kolbanovskaya E Y, Okorokov A L, Panova T B, Terwisscha van Scheltinga A C, Beintema J J
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
FEBS Lett. 1996 Nov 25;398(1):57-60. doi: 10.1016/s0014-5793(96)01173-8.
Bovine pancreatic ribonuclease A (RNase A) has been widely used as a convenient model for structural and functional studies. The enzyme catalyzes cleavage of phosphodiester bonds in RNA and related substrates. Three amino acid residues located at the active site of RNase A (His12, His119, and Lys41) are known to be involved in catalysis. Mutation of His119 to asparagine was generated to study the role of His119 in RNase A catalysis. The mutant enzyme has been isolated and characterized. The mutation significantly decreases the rate of the transesterification reaction and has no effect on substrate affinity of the enzyme. An analysis of the enzymatic properties of H119N RNase A suggests that the imidazole ring of His119 of the wild-type enzyme must be protonated in an enzyme-substrate productive complex. Thus our results indicate that a contribution of protonated His119 into the catalysis is not restricted to protonation of oxygen atom of the substrate leaving group and that His119 participates directly in a transition state stabilization via hydrogen bonding.
牛胰核糖核酸酶A（RNase A）已被广泛用作结构和功能研究的便捷模型。该酶催化RNA及相关底物中磷酸二酯键的裂解。已知位于RNase A活性位点的三个氨基酸残基（His12、His119和Lys41）参与催化作用。将His119突变为天冬酰胺以研究His119在RNase A催化中的作用。已分离并表征了突变酶。该突变显著降低了酯交换反应的速率，且对酶的底物亲和力没有影响。对H119N RNase A酶学性质的分析表明，野生型酶His119的咪唑环在酶 - 底物活性复合物中必须被质子化。因此，我们的结果表明，质子化的His119对催化作用的贡献不限于底物离去基团氧原子的质子化，并且His119通过氢键直接参与过渡态的稳定。
