エノルピルビルシキマート3リン酸塩 (EPSP) 合成酵素 (AroA) 催化EPSP水解の移行状態分析

Meiyan Lou¹, Steven K Burger, Meghann E Gilpin

¹Department of Chemistry & Chemical Biology, and †Department of Biochemistry & Biomedical Sciences, McMaster University , 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.

Journal of the American Chemical Society

|July 7, 2012

まとめ

炭素への陽子の移転の酵素触媒は困難である. エノルピルビルシキマート3リン酸合成酵素 (AroA) は,重要な中間物質を安定化するために早期の移行状態を使用して,この課題を克服します.

科学分野:

バイオケミストリーバイオケミストリー
酵素触媒は,酵素を触媒として用いる.
化学的運動学化学的運動学

背景:

炭素原子への陽子の移転は,高エネルギー障壁と不利な熱力学により,重要な触媒的障壁を提示します.
エノルピルビルシキマート3リン酸合成酵素 (EPSP合成酵素,AroA) は,その逆反応で,フォスフェノルピルバート (EPSP) のメチレン炭素をプロトン化するという課題に直面しています.

研究の目的:

酵素の逆反応のアナログとして機能するAroA触媒によるEPSP水解の移行状態を分析する.
メチレン炭素プロトネーションのメカニズムを調査し,AroAの触媒戦略を特定する.

主な方法:

トランジション状態の分析のために,3~14Cおよび溶媒デウテリウムKIEsを含む,動力同位体効果 (KIEs) を利用した.
どこにでも存在するリン酸濃度を管理するための無機リン酸浄化システムを開発しました.
移行状態の構造を分析し,実験データと比較するために計算モデリングを使用しました.

主要な成果:

反応は,不可逆的なC3プロトン化によって段階的に進行し,EPSPカチオン中間体 (AH(‡) *ANメカニズム) を形成します.
大規模な実験の3~14C KIE (1.032 ± 0.005) は,C3と陽子の運動との強い結合を示した.
非常に低い溶媒デウテリウムKIE (0.97 ± 0.04) が観察され,初期の移行状態と一致しています.
大きな2~14C KIEでは,Asp313とGlu341の"静電サンドイッチ"によるC2陽性電荷の安定化が示されました.

結論:

AroAは,酸触媒反応よりも早く移行状態をシフトさせ,重要なハモンドシフトを達成することによって,触媒を容易にします.
酵素の触媒戦略は,EPSPカチオン中間体の正電荷を安定させることです.
計算モデルにより,実験結果が裏付けられ,活性部位残留物のカタリシスにおける役割が強調された.

さらに関連する動画

13:05

Enzymatic Synthesis of Epoxidized Metabolites of Docosahexaenoic, Eicosapentaenoic, and Arachidonic Acids

Published on: June 28, 2019

08:34

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Published on: February 5, 2020

関連する概念動画

01:48

ATP Synthase: Mechanism

In animals, the mitochondrial F1F0 ATP synthase is the key protein that synthesizes ATP molecules through a complex catalytic mechanism. While the nuclear genome encodes the majority of ATP synthase subunits, the mitochondrial genome encodes some of the enzyme's most critical components. The formation of this multi-subunit enzyme is a complex multi-step process regulated at the level of transcription, translation, and assembly. Defects in one or more of these steps can result in decreased ATP...

02:24

Acid-Catalyzed Ring-Opening of Epoxides

Epoxides that are three-membered ring systems are more reactive than other cyclic and acyclic ethers. The high reactivity of epoxides originates from the strain present in the ring. This ring strain acts as a driving force for epoxides to undergo ring-opening reactions either with halogen acids or weak nucleophiles in the presence of mild acid. The acid catalyst converts the epoxide oxygen, a poor leaving group, into an oxonium ion, a better leaving group, making the reaction feasible. The...

01:31

ATP Energy Storage and Release

ATP is a highly unstable molecule. Unless quickly used to perform work, ATP spontaneously dissociates into ADP and inorganic phosphate (Pi), and the free energy released during this process is lost as heat. The energy released by ATP hydrolysis is used to perform work inside the cell and depends on a strategy called energy coupling. Cells couple the exergonic reaction of ATP hydrolysis with endergonic reactions, allowing them to proceed.
One example of energy coupling using ATP involves a...

02:45

E2 Reaction: Kinetics and Mechanism

SN2 substitutions and E2 eliminations of alkyl halides proceed via a concerted pathway. While the nucleophile attacks the alpha carbon in SN2 reactions, it functions as a strong base and abstracts a beta hydrogen in the E2 mechanism. The rate-limiting transition state in E2 elimination reactions is characterized by partially broken carbon–hydrogen and carbon–halogen bonds and a partially formed pi bond between the alpha and beta carbons. The beta hydrogen and halide are eliminated...

01:18

ATP Synthase: Structure

ATP synthase or ATPase is among the most conserved proteins found in bacteria, mammals, and plants. This enzyme can catalyze a forward reaction in response to the electrochemical gradient, producing ATP from ADP and inorganic phosphate. ATP synthase can also work in a reverse direction by hydrolyzing ATP and generating an electrochemical gradient. Different forms of ATP synthases have evolved special features to meet the specific demands of the cell. Based on their specific feature, ATP...

02:57

Sharpless Epoxidation

The conversion of allylic alcohols into epoxides using the chiral catalyst was discovered by K. Barry Sharpless and is known as Sharpless epoxidation. The use of a chiral catalyst enables the formation of one enantiomer of the product in excess. This chiral catalyst is mainly a chiral complex of titanium tetraisopropoxide and tartrate ester (specific stereoisomer). The stereoisomer used in the chiral catalyst dictates the formation of the enantiomer of the product. In other words, the use of...

関連する実験動画