Single-molecule kinetics reveals signatures of half-sites reactivity in dihydroorotate dehydrogenase A catalysis

Jue Shi, Joe Dertouzos, Ari Gafni, Duncan Steel, Bruce A. Palfey*

*Corresponding author for this work

Research output: Contribution to journalConference articlepeer-review

40 Citations (Scopus)

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% Intel-converting 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.

Original languageEnglish
Pages (from-to)5775-5780
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume103
Issue number15
DOIs
Publication statusPublished - 11 Apr 2006

Scopus Subject Areas

  • General

User-Defined Keywords

  • Cooperativity
  • Flavin
  • Pyrimidine

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