Lucas James E, Siegel Justin B
Genome Center, University of California, Davis, California, 95616.
Department of Chemistry, University of California, Davis, California, 95616.
Protein Sci. 2015 Jun;24(6):936-45. doi: 10.1002/pro.2669. Epub 2015 Apr 2.
Enzyme active site residues are often highly conserved, indicating a significant role in function. In this study we quantitate the functional contribution for all conserved molecular interactions occurring within a Michaelis complex for mannitol 2-dehydrogenase derived from Pseudomonas fluorescens (pfMDH). Through systematic mutagenesis of active site residues, we reveal that the molecular interactions in pfMDH mediated by highly conserved residues not directly involved in reaction chemistry can be as important to catalysis as those directly involved in the reaction chemistry. This quantitative analysis of the molecular interactions within the pfMDH active site provides direct insight into the functional role of each molecular interaction, several of which were unexpected based on canonical sequence conservation and structural analyses.
酶活性位点残基通常高度保守，这表明其在功能中起着重要作用。在本研究中，我们对荧光假单胞菌来源的甘露醇2-脱氢酶（pfMDH）的米氏复合物中发生的所有保守分子相互作用的功能贡献进行了定量分析。通过对活性位点残基进行系统诱变，我们发现，pfMDH中由不直接参与反应化学过程的高度保守残基介导的分子相互作用，对催化作用的重要性可能与直接参与反应化学过程的分子相互作用相当。对pfMDH活性位点内分子相互作用的这种定量分析，直接揭示了每种分子相互作用的功能作用，其中有几种基于经典序列保守性和结构分析是出乎意料的。
