期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2007
卷号：104
期号：9
页码：3153-3158
DOI：10.1073/pnas.0609103104
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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) x 104 and 1.8 ({+/-}0.4) x 104 s-1, respectively. These values compare to rate constants of {approx}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, {approx}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 IIMtl 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.
关键词：domain motion ; NMR ; protein dynamics ; relaxation dispersion ; phosphotransferase system
