Giraldo Jesús, De Maria Leonardo, Wodak Shoshana J
Grup de Modelització Estructural i Funcional de Sistemes Biològics, Institut de Neurociències and Unitat de Bioestadística, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain.
Proteins. 2004 Aug 1;56(2):261-76. doi: 10.1002/prot.20137.
The microbial ribonuclease barnase exhibits low catalytic activity toward GpN dinucleotides, where G is guanosine, p is phosphate and N represents any nucleoside. When a phosphate is added to the 3'-end, as in GpNp, substrate affinity is enhanced by one order of magnitude, and the catalytic rate by two. In order to gain insight into this phenomenon, we analyzed the nucleotide conformations and protein-nucleotide interactions of 4 ns molecular dynamics (MD) trajectories of complexes of barnase with guanylyl(3'-5') adenosine (GpA) and guanylyl(3'-5') adenosine 3'-monophosphate (GpAp), respectively, in the presence of solvent and counter ions. We found that, in a majority of the bound GpA conformations, the guanine base was firmly bound to the recognition site. The phosphate and adenosine moieties pointed into the solvent, and interactions with key catalytic residues were absent. In contrast, the bound GpAp adopted conformations in which all of the nucleotide portions remained tightly bound to the enzyme and interactions with key catalytic residues were maintained. These observations indicate that, for GpA, a significant proportion of the bound nucleotide adopts non-productive conformations and that adding the terminal phosphate as in GpAp shifts the equilibrium of the bound conformations towards structures capable of undergoing catalysis. Incorporating this property into the kinetic equations yields an increase in both the apparent rate constant (kcat) and the apparent dissociation constant (K(M)) for GpAp versus GpA. The increase in K(M), caused by the presence of additional non-productive binding modes for GpA, should however be counterbalanced by the propensity of free GpA to adopt folded conformations in solution, which are unable to bind the enzyme and by the tighter binding of GpAp (Giraldo J, Wodak SJ, Van Belle D. Conformational analysis of GpA and GpAp in aqueous solution by molecular dynamics and statistical methods. J Mol Biol 1998; 283:863-882). Addition of the terminal phosphate is shown to significantly influence the collective motion of the enzyme in a manner that fosters interactions with key catalytic residues, representing a further likely contribution to the catalytic rate enhancement.
微生物核糖核酸酶巴那斯酶对GpN二核苷酸（其中G是鸟苷，p是磷酸，N代表任何核苷）表现出较低的催化活性。当在3'端添加一个磷酸，如在GpNp中时，底物亲和力提高一个数量级，催化速率提高两个数量级。为了深入了解这一现象，我们分别分析了在有溶剂和抗衡离子存在的情况下，巴那斯酶与鸟苷酰（3'-5'）腺苷（GpA）和鸟苷酰（3'-5'）腺苷3'-单磷酸（GpAp）复合物的4纳秒分子动力学（MD）轨迹中的核苷酸构象和蛋白质-核苷酸相互作用。我们发现，在大多数结合的GpA构象中，鸟嘌呤碱基牢固地结合在识别位点上。磷酸和腺苷部分指向溶剂，并且不存在与关键催化残基的相互作用。相反，结合的GpAp采用的构象是所有核苷酸部分都与酶紧密结合，并保持与关键催化残基的相互作用。这些观察结果表明，对于GpA，很大一部分结合的核苷酸采用非生产性构象，并且如在GpAp中那样添加末端磷酸会使结合构象的平衡向能够进行催化的结构移动。将这一特性纳入动力学方程会使GpAp相对于GpA的表观速率常数（kcat）和表观解离常数（K(M)）都增加。然而，GpA存在额外的非生产性结合模式导致的K(M)增加，应该会被游离GpA在溶液中采用折叠构象（无法结合酶）的倾向以及GpAp更紧密的结合所抵消（吉拉尔多J、沃达克SJ、范贝勒D。通过分子动力学和统计方法对水溶液中GpA和GpAp的构象分析。《分子生物学杂志》1998年；283：863 - 882）。末端磷酸的添加被证明以促进与关键催化残基相互作用的方式显著影响酶的集体运动，这是催化速率提高的另一个可能原因。
