The role of glutamine 114 in Old Yellow Enzyme
The role of glutamine 114 in Old Yellow Enzyme
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DOI：
10.1074/jbc.m108453200
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发表时间：
2002-01-18
影响因子：
4.8
通讯作者：
Massey, V
中科院分区：
文献类型：
--
作者：
Brown, BJ;Hyun, JW;Massey, V
OYE1 的谷氨酰胺 114 是老黄酶家族活性位点中高度保守的残基。它与黄素蛋白辅基 FMN 的 02 和 N3 形成氢键。谷氨酰胺 114 突变为天冬酰胺，引入了一个短一个亚甲基的 R 基团。对所得酶进行表征以确定突变对酶机械行为的影响，并解析晶体结构以确定突变对蛋白质结构的影响。 Q114N 突变导致蛋白质结构几乎没有变化，将残基 114 的酰胺基移出氢键距离，从而允许 FAIN 辅基重新定位，形成新的相互作用来取代丢失的氢键。该突变降低了结合配体的能力，因为取代酚的所有解离常数都大于野生型酶。与 β-NADPH 发生还原半反应的速率常数稍大，而与 2-环己烯酮发生氧化半反应的速率常数则小于野生型酶。分子氧的氧化是双相的，涉及 O-2 的形成和反应，这种现象在这种突变中比野生型酶更明显。当超氧化物歧化酶添加到反应中时，我们观察到野生型酶典型的单相反应。研究了使用 β-NADPH 与 2-环己烯酮和分子氧的周转反应，以进一步表征突变酶。
Glutamine 114 of OYE1 is a well conserved residue in the active site of the Old Yellow Enzyme family. It forms hydrogen bonds to the 02 and N3 of the flavoprotein prosthetic group, FMN. Glutamine 114 was mutated to asparagine, introducing an R-group that is one methylene group shorter. The resultant enzyme was characterized to determine the effect of the mutation on the mechanistic behavior of the enzyme, and the crystal structure was solved to determine the effect of the mutation on the structure of the protein. The Q114N mutation results in little change in the protein structure, moving the amide group of residue 114 out of H-bonding distance, allowing repositioning of the FAIN prosthetic group to form new interactions that replace the lost H-bonds. The mutation decreases the ability to bind ligands, as all dissociation constants for substituted phenols are larger than for the wild type enzyme. The rate constant for the reductive half-reaction with beta-NADPH is slightly greater, whereas that for the oxidative half-reaction with 2-cyclohexenone is smaller than for the wild type enzyme. Oxidation with molecular oxygen is biphasic and involves formation and reaction with O-2, a phenomenon that is more pronounced with this mutation than with wild type enzyme. When superoxide dismutase is added to the reaction, we observe a single-phase reaction typical of the wild type enzyme. Turnover reactions using beta-NADPH with 2-cyclohexenone and molecular oxygen were studied to further characterize the mutant enzyme.