Doucet Nicolas, Watt Eric D, Loria J Patrick
Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA.
Biochemistry. 2009 Aug 4;48(30):7160-8. doi: 10.1021/bi900830g.
The role of the flexible loop 1 in protein conformational motion and in the dissociation of enzymatic product from ribonuclease A (RNase A) was investigated by creation of a chimeric enzyme in which a 6-residue loop 1 from the RNase A homologue, eosinophil cationic protein (ECP), replaced the 12-residue loop 1 in RNase A. The chimera (RNase A(ECP)) experiences only local perturbations in NMR backbone chemical shifts compared to WT RNase A. Many of the flexible residues that were previously identified in WT as involved in an important conformational change now experience no NMR-detected millisecond motions in the chimera. Likewise, binding of the product analogue, 3'-CMP, to RNase A(ECP) results in only minor chemical shift changes in the enzyme similar to what is observed for the H48A mutant of RNase A and in contrast to WT enzyme. For both RNase A(ECP) and H48A there is a 10-fold decrease in the product release rate constant, k(off), compared to WT, in agreement with previous studies indicating the importance of flexibility in RNase A in the overall rate-limiting product release step. Together, these NMR and biochemical experiments provide additional insight into the mechanism of millisecond motions in the RNase A catalytic cycle.
通过构建一种嵌合酶来研究柔性环1在蛋白质构象运动以及酶产物从核糖核酸酶A（RNase A）上解离过程中的作用。在该嵌合酶中，来自RNase A同源物嗜酸性粒细胞阳离子蛋白（ECP）的一个6残基环1取代了RNase A中的12残基环1。与野生型RNase A相比，该嵌合体（RNase A(ECP)）在核磁共振主链化学位移上仅经历局部扰动。许多先前在野生型中被确定参与重要构象变化的柔性残基，在嵌合体中现在未检测到核磁共振毫秒级运动。同样，产物类似物3'-CMP与RNase A(ECP)的结合，在酶中仅导致轻微的化学位移变化，这与在RNase A的H48A突变体中观察到的情况类似，与野生型酶形成对比。与野生型相比，RNase A(ECP)和H48A的产物释放速率常数k(off)均下降了10倍，这与先前研究一致，表明RNase A的柔性在总体限速产物释放步骤中具有重要性。这些核磁共振和生化实验共同为RNase A催化循环中毫秒级运动的机制提供了更多见解。
