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トリプシンをキモトリプシンに変換する: 表面ループの役割
L Hedstrom1, L Szilagyi, W J Rutter
1Hormone Research Institute, University of California, San Francisco 94143-0534.
Science (New York, N.Y.)
|March 6, 1992
まとめ
トリプシンを変化させる.
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:
- トリプシンとキモトリプシンは,構造が似ても基底特異性が異なるセリンタンパク質である.
- トリプシンは基本的残留物 (Arg,Lys) の後に分裂し,キモトリプシンは大きな水性残留物を標的とする.
- これらの特異性の違いを理解することで,特定の用途のための酵素工学に情報を与えることができます.
研究 の 目的:
- トリプシンとキモトリプシンにおける基板特異性の構造的決定因子を調査する.
- チモトリプシンのような基板特異性を持つトリプシン変異体を設計する.
- 酵素触媒と差別化におけるS1結合部位と表面ループの役割を明らかにする.
主な方法:
- サイト指向型変異は,トリプシンのS1結合部位をキモトリプシン残留物で置き換えるために使用されました.
- さらに,トリプシンとキモトリプシンとの間の表面ループの交換 (残留185-188および221-225) も行われました.
- 酵素動力学および基質結合アッセイは,野生型および変異性酵素で実施されました.
主要な成果:
- S1部位のみを置換すると,エステラーゼ特異性が伝わり,アミド水解特異性は伝わりませんでした.
- S1サイトと特定の表面ループの両方に変異を加え,トリプシンをキモトリプシンのような酵素に変換した.
- エンジニアリングされた突然変異体はキモトリプシンの触媒率を示したが,基質結合が低下していた.
- 変異体における基質の差別は,アシル化中に発生し,キモトリプシンと類似して結合しない.
結論:
- 活性部位に直接含まれていない表面ループは,プロテアゼ基板特異性に大きく影響する.
- 酵素特異性は,活性部位と遠隔構造要素の複雑な相互作用である.
- この研究は,新しい触媒特性を持つ酵素を作成するためのタンパク質工学に関する洞察を提供します.
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