| Conformational events during ternary enzyme-substrate complex formation are rate limiting in the catalytic cycle of the light-driven enzyme protochlorophyllide oxidoreductase. | |
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MedLine Citation:
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PMID: 18798649 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The light-driven enzyme, protochlorophyllide oxidoreductase (POR), has proven to be an excellent model system for studying the role of protein motions during catalysis. POR catalyzes the trans addition of hydrogen across the C17-C18 double bond of protochlorophyllide (Pchlide), which is a key step in chlorophyll biosynthesis. While we currently have a detailed understanding of the initial photochemical events and the subsequent hydrogen transfer reactions, there remains a lack of information about the slower substrate binding events leading to the formation of the catalytically active ternary complex. As POR is light-activated, it is relatively straightforward to isolate the ternary enzyme-substrate complex in the dark prior to catalysis, which has facilitated the use of a variety of spectroscopic and kinetic probes to study the binding of both substrates. Herein, we provide a detailed kinetic and thermodynamic description of these processes and show that the binding events are complex, involving multiple conformational states en route to the formation of a ternary complex that is primed for photoactivation. The initial binding of NADPH involves three distinct steps, which appear to be necessary for the optimal alignment of the cofactor in the enzyme active site. This is followed by the binding of the Pchlide substrate and subsequent substrate-induced conformational changes within the enzyme that occur prior to the formation of the final "poised" conformational state. These studies, which provide important information on the formation of the reactive conformation, reveal that ternary complex formation is the rate-limiting step in the overall reaction and is controlled by slow conformational changes in the protein. |
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Authors:
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Derren J Heyes; Binuraj R K Menon; Michiyo Sakuma; Nigel S Scrutton |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2008-09-18 |
Journal Detail:
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Title: Biochemistry Volume: 47 ISSN: 1520-4995 ISO Abbreviation: Biochemistry Publication Date: 2008 Oct |
Date Detail:
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Created Date: 2008-10-07 Completed Date: 2008-10-28 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 0370623 Medline TA: Biochemistry Country: United States |
Other Details:
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Languages: eng Pagination: 10991-8 Citation Subset: IM |
Affiliation:
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Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK. derren.heyes@manchester.ac.uk |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Catalysis Fluorescence Resonance Energy Transfer Kinetics Light* NADP / metabolism Oxidoreductases Acting on CH-CH Group Donors / metabolism* Protein Binding Protein Conformation Spectroscopy, Fourier Transform Infrared Substrate Specificity |
| Grant Support | |
ID/Acronym/Agency:
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//Biotechnology and Biological Sciences Research Council |
| Chemical | |
Reg. No./Substance:
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53-59-8/NADP; EC 1.3.-/Oxidoreductases Acting on CH-CH Group Donors; EC 1.3.1.33/protochlorophyllide reductase |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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