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Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope
Published on: February 28, 2019
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Subnanometre enzyme mechanics probed by single-molecule force spectroscopy.
Benjamin Pelz1, Gabriel Žoldák1, Fabian Zeller2
1Physik Department E22, Technische Universität München, James Franck Strasse 1, 85748 Garching, Germany.
Nature Communications
|February 25, 2016
Summary
Enzymes like adenylate kinase use lid closing to function. Substrate binding surprisingly favors a half-closed state, revealing new insights into enzyme mechanics and efficient catalysis.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Enzymes are critical molecular machines facilitating biochemical reactions through specific substrate binding and catalysis.
- Understanding the energetic forces driving enzyme conformational changes, particularly lid closing, is essential for elucidating catalytic mechanisms.
- Adenylate kinase (AK) is a key enzyme involved in energy metabolism, featuring mobile lid domains crucial for its function.
Purpose of the Study:
- To investigate the energetic landscape of substrate-dependent lid conformational changes in adenylate kinase.
- To elucidate the mechanical coupling between substrate/inhibitor binding and enzyme lid dynamics.
- To provide atomic-level insights into how enzymes balance substrate exchange efficiency with catalytic site closure.
Main Methods:
- Utilized sub-nanometer single-molecule force spectroscopy (SMFS) to precisely measure forces and energetics during enzyme conformational changes.
- Employed molecular dynamics (MD) simulations to complement experimental data and provide detailed atomic-level mechanistic insights.
- Studied the effects of a bisubstrate inhibitor (diadenosine pentaphosphate, AP5A) and substrates (ADP, ATP) on adenylate kinase lid dynamics.
Main Results:
- In the presence of the inhibitor AP5A, adenylate kinase lid opening and closing are cooperative and tightly coupled to inhibitor binding.
- Substrate binding (ADP and ATP) exhibits a significantly weaker energetic drive towards the fully closed state compared to the inhibitor.
- A novel, dominant energetic minimum was identified with both enzyme lids in a half-closed conformation upon substrate binding.
Conclusions:
- Enzyme lid dynamics are finely tuned by the nature of the bound ligand, with inhibitors promoting full closure and substrates favoring an intermediate state.
- The observed half-closed state represents a crucial mechanical intermediate, balancing the need for rapid substrate/product exchange with maintaining catalytic efficiency.
- This study provides unprecedented mechanical insights into the complex interplay of forces governing enzyme function and catalysis.
