Single-Molecule Kinetics Reveals Signatures of Half-Sites Reactivity in Dihydroorotate Dehydrogenase a Catalysis

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 enz...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2006-04, Vol.103 (15), p.5775-5780
Main Authors: Shi, Jue, Dertouzos, Joe, Gafni, Ari, Steel, Duncan, Palfey, Bruce A.
Format: Article
Language:English
Subjects:
Bacteria
Bacterial Proteins - chemistry
Bacterial Proteins - metabolism
Binding Sites
Biological Sciences
Biophysics
Catalysis
Dehydrogenases
Dimerization
Dimers
Enzymes
Flavin Mononucleotide - metabolism
Fluorescence
Kinetics
Lactococcus lactis
Lactococcus lactis - enzymology
Models, Theoretical
Molecules
Oxidation
Oxidoreductases Acting on CH-CH Group Donors - chemistry
Oxidoreductases Acting on CH-CH Group Donors - metabolism
Protein Subunits - chemistry
Protein Subunits - metabolism
Reactivity
Trajectories
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0510482103