Intramolecular domain-domain association/dissociation and phosphoryl transfer in the mannitol transporter of Escherichia coli are not coupled

Jeong Yong Suh, Junji Iwahara, G. Marius Clore

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

The Escherichia coli mannitol transporter (IIMtl) comprises three domains connected by flexible linkers: a transmembrane domain (C) and two cytoplasmic domains (A and B). IIMtl catalyzes three successive phosphoryl-transfer reactions: one intermolecular (from histidine phosphocarrier protein to the A domain) and two intramolecular (from the A to the B domain and from the B domain to the incoming sugar bound to the C domain). A key functional requirement of IIMtl is that the A and B cytoplasmic domains be able to rapidly associate and dissociate while maintaining reasonably high occupancy of an active stereospecific AB complex to ensure effective phosphoryl transfer along the pathway. We have investigated the rate of intramolecular domain-domain association and dissociation in IIBAMtl by using 1H relaxation dispersion spectroscopy in the rotating frame. The open, dissociated state (comprising an ensemble of states) and the closed, associated state (comprising the stereospecific complex) are approximately equally populated. The first-order rate constants for intramolecular association and dissociation are 1.7 (±0.3) × 104 and 1.8 (±0.4) × 104 s-1, respectively. These values compare to rate constants of ≈500 s-1 for A → B and B → A phosphoryl transfer, derived from qualitative line-shape analysis of 1H-15N correlation spectra taken during the course of active catalysis. Thus, on average, ≈80 association/dissociation events are required to effect a single phosphoryl-transfer reaction. We conclude that intramolecular phosphoryl transfer between the A and B domains of II Mtl is rate-limited by chemistry and not by the rate of formation or dissociation of a stereospecific complex in which the active sites are optimally apposed.

Original languageEnglish (US)
Pages (from-to)3153-3158
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number9
DOIs
StatePublished - Feb 27 2007

Keywords

  • Domain motion
  • NMR
  • Phosphotransferase system
  • Protein dynamics
  • Relaxation dispersion

ASJC Scopus subject areas

  • General

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