A simple method to resolve rate constants when the binding mechanism obeys induced fit or conformational selection
Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved..
Many interactions involving a ligand and its molecular target are studied by rapid kinetics using a stopped-flow apparatus. Information obtained from these studies is often limited to a single, saturable relaxation that is insufficient to resolve all independent rate constants even for a two-step mechanism of binding obeying induced fit (IF) or conformational selection (CS). We introduce a simple method of general applicability where this limitation is overcome. The method accurately reproduces the rate constants for ligand binding to the serine protease thrombin determined independently from the analysis of multiple relaxations. Application to the inactive zymogen precursor of thrombin, prethrombin-2, resolves all rate constants for a binding mechanism of IF or CS from a single, saturable relaxation. Comparison with thrombin shows that the prethrombin-2 to thrombin conversion enhances ligand binding to the active site not by improving accessibility through the value of kon but by reducing the rate of dissociation koff. The conclusion holds regardless of whether binding is interpreted in terms of IF or CS and has general relevance for the mechanism of zymogen activation of serine proteases. The method also provides a simple test of the validity of IF and CS and indicates when more complex mechanisms of binding should be considered.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:300 |
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| Enthalten in: |
The Journal of biological chemistry - 300(2024), 4 vom: 27. Apr., Seite 107131 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Di Cera, Enrico [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 27.04.2024 Date Revised 27.04.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.jbc.2024.107131 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM369225082 |
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| 100 | 1 | |a Di Cera, Enrico |e verfasserin |4 aut | |
| 245 | 1 | 2 | |a A simple method to resolve rate constants when the binding mechanism obeys induced fit or conformational selection |
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| 500 | |a Date Completed 27.04.2024 | ||
| 500 | |a Date Revised 27.04.2024 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved. | ||
| 520 | |a Many interactions involving a ligand and its molecular target are studied by rapid kinetics using a stopped-flow apparatus. Information obtained from these studies is often limited to a single, saturable relaxation that is insufficient to resolve all independent rate constants even for a two-step mechanism of binding obeying induced fit (IF) or conformational selection (CS). We introduce a simple method of general applicability where this limitation is overcome. The method accurately reproduces the rate constants for ligand binding to the serine protease thrombin determined independently from the analysis of multiple relaxations. Application to the inactive zymogen precursor of thrombin, prethrombin-2, resolves all rate constants for a binding mechanism of IF or CS from a single, saturable relaxation. Comparison with thrombin shows that the prethrombin-2 to thrombin conversion enhances ligand binding to the active site not by improving accessibility through the value of kon but by reducing the rate of dissociation koff. The conclusion holds regardless of whether binding is interpreted in terms of IF or CS and has general relevance for the mechanism of zymogen activation of serine proteases. The method also provides a simple test of the validity of IF and CS and indicates when more complex mechanisms of binding should be considered | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, N.I.H., Extramural | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a enzyme mechanism | |
| 650 | 4 | |a presteady-state kinetics | |
| 650 | 4 | |a prothrombin | |
| 650 | 4 | |a serine protease | |
| 650 | 4 | |a thrombin | |
| 650 | 7 | |a Thrombin |2 NLM | |
| 650 | 7 | |a EC 3.4.21.5 |2 NLM | |
| 650 | 7 | |a Ligands |2 NLM | |
| 650 | 7 | |a Enzyme Precursors |2 NLM | |
| 650 | 7 | |a Prothrombin |2 NLM | |
| 650 | 7 | |a 9001-26-7 |2 NLM | |
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