Cation-promoted association of Escherichia coli phosphocarrier protein IIAGlc with regulatory target protein glycerol kinase: substitutions of a Zinc(II) ligand and implications for inducer exclusion.
In Escherichia coli, inducer exclusion is one mechanism by which glucose prevents unnecessary expression of genes needed for metabolism of other sugars. The basis for this mechanism is binding of the unphosphorylated form of the glucose-specific phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system, IIAGlc (also known as IIIGlc), to a variety of target proteins to prevent uptake or synthesis of the inducer. One of these target proteins is glycerol kinase (EC 2.1.7.30, ATP:glycerol 3-phosphotransferase), which is inhibited by IIAGlc. Glycerol kinase is the only IIAGlc target protein for which the structure of the complex is known. Association of these two proteins forms an intermolecular binding site for Zn(II) with metal ligands contributed by each protein, and Zn(II) enhances IIAGlc inhibition [Feese, M., Pettigrew, D. W., Meadow, N. D., Roseman, S., and Remington, S. J. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3544-3548]. Here, we show that the Zn(II) enhancement can be described quantitatively by a model with binding of Zn(II) to the complex with an apparent dissociation constant of less than 1 microM at pH 7.0 and 25 degreesC. Initial velocity studies show that IIAGlc is an uncompetitive inhibitor with respect to both substrates, and the mechanism of inhibition is not altered by Zn(II). The Zn(II)-liganding residue contributed by glycerol kinase (Glu478) is substituted by using site-directed mutagenesis to construct the enzymes E478C, E478D, E478H, and E478Q. The substitutions have only small effects on the inhibition by IIAGlc in the absence of Zn(II), the catalytic properties, or other allosteric regulation. However, all of the substitutions abolish the Zn(II) enhancement of IIAGlc inhibition, and the X-ray crystallographic structures of the complexes of IIAGlc with the E478C and E478H mutants show these substitutions abolish binding of Zn(II) to the intermolecular site. These results support the hypothesis that Zn(II) enhances the affinity for complex formation by binding at the intermolecular site, i.e., cation promoted association. The high affinity for Zn(II) binding to the complex and the ability of the other four amino acid residues to efficiently substitute for Glu478 in all functions except binding of Zn(II) suggest that cation promoted association of these two proteins may have a role in inducer exclusion in vivo.
Pettigrew
D W
DW
Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Meadow
N D
ND
Roseman
S
S
Remington
S J
SJ
D002412:Cations; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005991:Glycerol Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D002412:Cations; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C032242:crr protein, E coli; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D005991:Glycerol Kinase; D015032:Zinc