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 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.
Biophysics Research Division and Department of Physics, University of Michigan, Ann Arbor, MI 48109, USA.
Jue
J
Shi
Joe
J
Dertouzos
Ari
A
Gafni
Duncan
D
Steel
Bruce A
BA
Palfey
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D000090722:Dihydroorotate Dehydrogenase; D019281:Dimerization; D005486:Flavin Mononucleotide / Q000378:metabolism; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology; D008962:Models, Theoretical; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D000090722:Dihydroorotate Dehydrogenase; D021122:Protein Subunits; D005486:Flavin Mononucleotide; D044925:Oxidoreductases Acting on CH-CH Group Donors