ArticleProceedings of the National Academy of Sciences of the United States of America2006
Single-molecule kinetics reveals signatures of half-sites reactivity in dihydroorotate dehydrogenase A catalysis.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2006. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 45 citations in OpenAlex.
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- Shedding light on protein folding, structural and functional dynamics by single molecule studies.Molecules (Basel, Switzerland) · 2014Review
- Single-Molecule Fluorescence Using Nucleotide Analogs: A Proof-of-Principle.The journal of physical chemistry letters · 2014Article
- 1,4-naphthoquinones and other NADPH-dependent glutathione reductase-catalyzed redox cyclers as antimalarial agents.Current pharmaceutical design · 2013Review
- Observation of protein folding/unfolding dynamics of ubiquitin trapped in agarose gel by single-molecule FRET.European biophysics journal : EBJ · 2012Article
- Single-molecule analysis reveals three phases of DNA degradation by an exonuclease.Nature chemical biology · 2011Article
- Metallic-Nanostructure-Enhanced Fluorescence of Single Flavin Cofactor and Single Flavoenzyme Molecules.The journal of physical chemistry. C, Nanomaterials and interfaces · 2011Article
- Rate theories for biologists.Quarterly reviews of biophysics · 2010Review
- Probing biomolecular structures and dynamics of single molecules using in-gel alternating-laser excitation.Analytical chemistry · 2009Article
- Determination of the oligomer size of amyloidogenic protein beta-amyloid(1-40) by single-molecule spectroscopy.Biophysical journal · 2009Article
- Structural evidence for substrate-induced synergism and half-sites reactivity in biotin carboxylase.Protein science : a publication of the Protein Society · 2008Article
- The enzyme mechanism of nitrite reductase studied at single-molecule level.Proceedings of the National Academy of Sciences of the United States of America · 2008Article
Corrections and comments
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
Abstract
Subunit activity and cooperativity of a homodimeric flavoenzyme, dihydroorotate dehydrogenase A (DHODA) from Lactococcus lactis, were characterized by employing single-molecule spectroscopy to follow the turnover kinetics of individual DHODA molecules, eliminating ensemble averaging. Because the enzyme-bound FMN is fluorescent in its oxidized state but not when reduced, a single DHODA molecule exhibits stepwise fluorescence changes during turnover, providing a signal to determine reaction kinetics and study cooperativity. Our results showed significant heterogeneity in the catalytic behaviors of individual dimer molecules, with only 40% interconverting between the three possible redox states: the fully fluorescent (both subunits oxidized), the half-fluorescent (one subunit oxidized and the other reduced), and the nonfluorescent (both subunits reduced). Forty percent of the single dimer traces showed turnovers between only the fully fluorescent and half-fluorescent states. The remaining 20% of the molecules interconverted only between the half-fluorescent state and the nonfluorescent state. Kinetic analysis revealed very similar reaction rates in both the reductive and oxidative half-reactions for different DHODA dimers. Our single-molecule data provide strong evidence for half-sites reactivity, in which only one subunit reacts at a time. The present study presents an effective way to explore the subunit catalytic activity and cooperativity of oligomeric enzymes by virtue of single-molecule fluorescence.
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