酵素構造:トリオセフォスファートイソメラーゼの活性部位グルタミン酸の基本性を最適化する機能を持つアミノ酸サイドチェーン
Xiang Zhai1, Christopher J Reinhardt2, M Merced Malabanan3
1Department of Chemistry , University at Buffalo, SUNY , Buffalo , New York 14260-3000 United States.
Journal of the American Chemical Society
|June 5, 2018
まとめ
この研究では,トリオフォスファートイソメラーゼ (TIM) 酵素の運動と,フォスフォグリコラートトリアニオンによる抑制を調査しています. TIM を変化させる変異
科学分野:
- 生物化学
- 酵素運動
- タンパク質工学
背景:
- トリオフォスファートイソメラーゼ (TIM) は,糖分解の重要なステップを触媒化する.
- TIMの触媒メカニズムと基板結合を理解することは,代謝経路の研究に不可欠です.
- 酵素活性部位の相互作用に関する洞察を,フォスフォグリコラトトライアニオンなどの阻害剤が提供します.
研究 の 目的:
- ワイルドタイプと突然変異のTIMのpH依存運動を特徴付ける.
- TIMの触媒活性と基板結合における特定のアミノ酸残基の役割を明らかにする.
- フォスフォグリコラトトライアニオンによる抑制メカニズムを調査する.
主な方法:
- kcatとKmを測定する酵素運動測定法
- フォスフォグリコラートトリアニオンの抑制定数 (Ki) の決定
- 野生型と変異型TIMのpH率プロフィールの分析
- 線形自由エネルギー相関分析
主要な成果:
- pHプロファイルは,TIMへのフォスフォグリコラトトライアニオン結合中のプロトネーションイベントを明らかにします.
- 触媒効率 (kcat/Km) と阻害剤結合親和性との間に強い相関が認められた.
- 変異は重要な活性部位残基の触媒活性とpKaの両方を有意に変化させた.
- 特定の残留物 (E165,Y208,S211,I170,P166) は,TIMの機能にとって重要であると特定されました.
結論:
- この研究は,特定のアミノ酸残留が移行状態を安定させ,E165の基本性を高めるモデルを支持している.
- これらの残基に影響する変異は,触媒効率と阻害剤結合の両方に影響を及ぼします.
- TIMの触媒メカニズムは,基質イソメリゼーションを容易にするために,活性サイト残基の正確な位置づけを含みます.
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