Department of Biochemistry and Biophysics, Texas A&M University, College Station, 77843, USA.
Bioorg Chem. 2010 Jun;38(3):115-9. doi: 10.1016/j.bioorg.2009.12.004. Epub 2009 Dec 28.
The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Ser171 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by approximately 5-fold and decreases in the rate constant for product release of approximately 2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure.
黄素酶硝基烷氧化酶催化伯硝基烷和仲硝基烷氧化为相应的醛和酮以及亚硝酸盐。该酶的结构表明，Ser171 与黄素 N5 形成氢键，表明其在催化中起作用。Cys397 和 Tyr398 先前通过化学修饰被鉴定为潜在的活性位点残基。为了更直接地探究这些残基的作用，已经对 S171A、S171V、S171T、C397S 和 Y398F 酶进行了以硝基乙烷为底物的表征。C397S 和 Y398 酶比野生型酶的稳定性差，并且 C397S 酶通常含有亚化学计量的 FAD。对突变酶的稳态动力学参数进行分析，包括氘同位素效应，确定所有突变都导致去除底物质子的速率常数降低约 5 倍，产物释放的速率常数降低约 2 倍。只有 S171V 和 S171T 突变改变了黄素氧化的速率常数。这些结果表明这些残基不参与催化，而是维持蛋白质结构所必需的。
