活性部位であるフェニララニンは,アルファケトグルタレート依存型ダイオキシゲナーゼ TauD の基板結合とC-H分裂を誘導する

Kevin P McCusker¹, Judith P Klinman

¹Department of Chemistry, California Institute for Quantitative Biosciences, University of California, Berkeley, California 94720, USA.

Journal of the American Chemical Society

|March 23, 2010

まとめ

タウリンアルファケトグルタレート酸化酵素 (TauD) のフェニララニン159は,基板結合とC-H結合の分裂に不可欠です. 変異は最適な距離を乱し,酵素の効率と水素波関数の重複に影響を与えます.

科学分野:

バイオケミストリーバイオケミストリー
酵素学酵素学とは
プロテイン工学は,タンパク質の

背景:

C-H結合を分裂する酵素は,しばしば,触媒作用のための基板を配置するために,水嫌性核に依存します.
タウリンアルファ-ケトグルタ酸二酸化酵素 (TauD) は,C-H結合の酸化のために活性サイトFe(IV) = O種を使用します.
TauDの残留物F159は,水素原子移転の基板結合と指向に関与しています.

研究の目的:

TauDの触媒機構における残留物F159の役割を調査する.
F159の突然変異が基質結合,C-H結合分裂,酸素活性化にどのように影響するかを決定する.
活性部位構造,基板方向,および酵素効率の間の関係を調査する.

主な方法:

TauDにおけるF159のサイト指向型変異は,より小さな水性残留物 (L,V,A) に変異する.
定常状態の回転条件下での酵素運動学的研究.
シングル・ターンオーバー・トランジントを用いた固有デウテリウム運動同位体効果 (KIE) の抽出.

主要な成果:

F159における突然変異は,基質結合とC−H結合の割裂率を著しく低下させた.
k ((cat)) に対する化学ステップの貢献は,F159変異体において増加し,変化した速度制限ステップを示した.
固有のデウテリウムKIEは,野生型とF159Lと比較してF159Vの2〜4倍に増加しました.
F159LとF159Vの変異の間に,酵素の行動の明確な変化が観察されました.

結論:

残留F159は,基板とFe(IV) = O活性部位との間の最適な核間距離を維持するために重要である.
F159Vにおける単一のメチレン基の喪失は,効率的な水素原子移転を促進する活性部位の能力を破壊する.
この発見は,TauDのモジュラー型活性部位モデルを支持し,基板結合/指向と酸素活性化のための明確な機能的役割を有する.

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