The glucose‐specific carrier of the <i>Escherichia coli</i> phosphotransferase system

Garcia-Alles, Luis F.; Navdaeva, Vera; Haenni, Simon; Erni, Bernhard

doi:10.1046/j.1432-1033.2002.03197.x

Cited by 11 publications

(6 citation statements)

References 45 publications

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“…(EII glc ), a model has been proposed with both a high-and a low-affinity binding site (49), but direct evidence for this is lacking. Although EII mtl and EII glc are not homologous, they behave catalytically in a similar manner and we feel that a reevaluation of the number of substrate binding sites in EII glc may be necessary.…”

Section: Discussionmentioning

confidence: 99%

Stoichiometry and Substrate Affinity of the Mannitol Transporter, EnzymeIImtl, from Escherichia coli

Veldhuis

Broos

Poolman

et al. 2005

Biophysical Journal

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Uptake and consecutive phosphorylation of mannitol in Escherichia coli is catalyzed by the mannitol permease EnzymeIImtl. The substrate is bound at an extracellular-oriented binding site, translocated to an inward-facing site, from where it is phosphorylated, and subsequently released into the cell. Previous studies have shown the presence of both a high- and a low-affinity binding site with K(D)-values in the nano- and micromolar range, respectively. However, reported K(D)-values in literature are highly variable, which casts doubts about the reliability of the measurements and data analysis. Using an optimized binding measurement system, we investigated the discrepancies reported in literature, regarding both the variability in K(D)-values and the binding stoichiometry. By comparing the binding capacity obtained with flow dialysis with different methods to determine the protein concentration (UV-protein absorption, Bradford protein detection, and a LDH-linked protein assay to quantify the number of phosphorylation sites), we proved the existence of only one mannitol binding site per dimeric species of unphosphorylated EnzymeIImtl. Furthermore, the affinity of EnzymeIImtl for mannitol appeared to be dependent on the protein concentration and seemed to reflect the presence of an endogenous ligand. The dependency could be simulated assuming that >50% of the binding sites were occupied with a ligand that shows an affinity for EnzymeIImtl in the same range as mannitol.

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Section: Discussionmentioning

confidence: 99%

Stoichiometry and Substrate Affinity of the Mannitol Transporter, EnzymeIImtl, from Escherichia coli

Veldhuis

Broos

Poolman

et al. 2005

Biophysical Journal

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“…Garcia-Alles et al (18,33) reported that IICB Glc from E. coli exhibits steady-state kinetics that are biphasic when a large range of sugar concentrations (50 M to 5 mM) is tested. The authors attribute this to the presence of at least three (and perhaps four) catalytic sites that fall into two classes, one with higher affinity for Glc (K S ϳ 10 M) but lower phosphorylation activity and the other with low affinity for Glc (K S ϳ300 M) but about 6 times the phosphorylation activity of the high affinity class.…”

Section: Discussionmentioning

confidence: 99%

Transient State Kinetics of Enzyme IICBGlc, a Glucose Transporter of the Phosphoenolpyruvate Phosphotransferase System of Escherichia coli

Meadow

Savtchenko

Nezami³

et al. 2005

Journal of Biological Chemistry

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During translocation across the cytoplasmic membrane of Escherichia coli, glucose is phosphorylated by phospho-IIA Glc and Enzyme IICB Glc , the last two proteins in the phosphotransfer sequence of the phosphoenolpyruvate:glucose phosphotransferase system. Transient state (rapid quench) methods were used to determine the second order rate constants that describe the phosphotransfer reactions (phospho-IIA Glc to IICB Glc to Glc) and also the second order rate constants for the transfer from phospho-IIA to the phosphorylation site Cys of IIB Glc or IICB Glc were found to be 3.5 and 12, respectively. These equilibrium constants signify that the thiophospho-group in these proteins has a high phosphotransfer potential, similar to that of the phosphohistidinyl phosphotransferase system proteins. In these studies, preparations of IICB Glc were invariably found to contain endogenous, firmly bound Glc (estimated K D ϳ10 ؊7 M). The bound Glc was kinetically competent and was rapidly phosphorylated, indicating that IICB Glc has a random order, Bi Bi, substituted enzyme mechanism. The equilibrium constant for the binding of Glc was deduced from differences in the statistical goodness of fit of the phosphotransfer data to the kinetic model.The bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) 3 comprises dozens of cytoplasmic and membrane proteins, most of which are the sugar-specific components of the system (for reviews, see Refs. 1-4). The PTS has several important functions in eubacterial cell physiology in addition to its major role in sugar transport, especially in Gram-positive bacteria (5).When the PTS catalyzes sugar transport, the sugar is phosphorylated as it crosses the membrane, and this process requires 3-6 proteins, depending on the sugar. One example is the Glc transport system in enteric bacteria, shown in Fig. 1.The first two proteins, Enzyme I and HPr, are the so-called general proteins of the system, meaning that they are not sugar-specific. The second pair of proteins, IIA Glc and IICB Glc , are the sugar-specific components. In other cases, the sugar-specific proteins vary from one to four separately encoded proteins. All of the phosphotransfer reactions except the last step (transfer to the sugar) are physiologically reversible. A second point is that the phosphorylation site residues in the PTS proteins are generally His, but in a number of the membrane proteins, the phosphorylation site can be a Cys residue. The phosphoryl group is transferred from PEP through a chain of His proteins to the membrane Enzyme II, where the phosphorylation site can be a His or a Cys (as it is in IICB Glc ), and finally to the sugar. In IICB Glc , the B domain extends into the cytoplasm and contains the phosphorylation site Cys.Our long term goal is to be able to predict the kinetics of Glc uptake by intact cells, and for this purpose it is necessary to obtain the rate constants for each of the reactions in Fig. 1. We established the basic methodology by developing a rapid quench method for determini...

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“…Initial kinetic analysis of both PtsG and MtlA demonstrated biphasic behavior, suggesting the presence of multiple binding sites with differing affinities[49, 64-66]. A more recent study concluded that MtlA has only one high affinity binding site per dimer (K D ≈ 100 nM) and that variations in the binding affinity constant were caused by contamination from an endogenous ligand[67].…”

Section: Transportmentioning

confidence: 99%

Structural insight into the PTS sugar transporter EIIC

McCoy

Levin

Zhou

2015

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background The enzyme IIC component (EIIC) of the phosphotransferase system (PTS) is responsible for selectively transporting sugar molecules across the inner bacterial membrane. This is accomplished in parallel with phosphorylation of the sugar, which prevents efflux of the sugar back across the membrane. This process is a key part of an extensive signaling network that allows bacteria to efficiently utilize preferred carbohydrate sources. Scope of review The goal of this review is to examine the current understanding of the structural features of EIIC and how it mediates concentrative, selective sugar transport. The crystal structure of an N,N’-diacetylchitobiose transporter is used as a structural template for the glucose superfamily of PTS transporters. Major conclusions Comparison of protein sequences in context with the known EIIC structure suggests members of the glucose superfamily of PTS transporters may exhibit variations in topology. Despite these differences, a conserved histidine and glutamate appear to have roles shared across the superfamily in sugar binding and phosphorylation. In the proposed transport model, a rigid body motion between two structural domains and movement of an intracellular loop provide the substrate binding site with alternating access, and reveal a surface required for interaction with the phosphotransfer protein required for catalysis. General significance The structural and functional data discussed here give a preliminary understanding of how transport in EIIC is achieved. However, given the great sequence diversity between varying glucose-superfamily PTS transporters and lack of data on conformational changes needed for transport, additional structures of other members and conformations are still required.

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The glucose‐specific carrier of the Escherichia coli phosphotransferase system

Cited by 11 publications

References 45 publications

Stoichiometry and Substrate Affinity of the Mannitol Transporter, EnzymeIImtl, from Escherichia coli

Stoichiometry and Substrate Affinity of the Mannitol Transporter, EnzymeIImtl, from Escherichia coli

Transient State Kinetics of Enzyme IICBGlc, a Glucose Transporter of the Phosphoenolpyruvate Phosphotransferase System of Escherichia coli

Structural insight into the PTS sugar transporter EIIC

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