CATION-PROMOTED ASSOCIATION OF ESCHERICHIA-COLI PHOSPHOCARRIER PROTEIN IIA(GLC) 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 gluco se prevents unnecessary expression of genes needed for metabolism of o ther sugars. The basis for this mechanism is binding of the unphosphor ylated form of the glucose-specific phosphocarrier protein of the phos phoenolpyruvate:glycose phosphotransferase system, IIA(Glc) (also know n as IIIGlc), to a variety of target proteins to prevent uptake or syn thesis of the inducer. One of these target proteins is glycerol kinase (EC 2.1.7.30, ATP:glycerol 3-phosphotransferase), which is inhibited by IIA(Glc). Glycerol kinase is the only IIA(Glc) target protein for w hich the structure of the complex is known. Association of these two p roteins forms an intermolecular binding site for Zn(II) with metal lig ands contributed by each protein, and Zn(II) enhances IIA(Glc) inhibit ion [Feese, M., Pettigrew, D. W., Meadow, N. D., Roseman, S., and Remi ngton, 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 diss ociation constant of less than 1 mu M at pH 7.0 and 25 degrees C. Init ial velocity studies show that IIA(Glc) is an uncompetitive inhibitor with respect to both substrates, and the mechanism of inhibition is no t altered by Zn(II). The Zn(II)-liganding residue contributed by glyce rol kinase (Glu478) is substituted by using site-directed mutagenesis to construct the enzymes E478C, E478D, E478H, and E478Q. The substitut ions have only small effects on the inhibition by IIA(Glc) in the abse nce of Zn(II), the catalytic properties, or other allosteric regulatio n. However, all of the substitutions abolish the Zn(II) enhancement of IIA(Glc) inhibition, and the X-ray crystallographic structures of the complexes of IIA(Glc) with the E478C and E478H mutants show these sub stitutions 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 comp lex and the ability of the other four amino acid residues to efficient ly substitute for Glu478 in all functions except binding of Zn(II) sug gest that cation promoted association of these two proteins may have a role in inducer exclusion in vivo.