Amino acid substitutions in the sugar kinase/hsp70/actin superfamily conserved ATPase core of E. coli glycerol kinase modulate allosteric ligand affinity but do not alter allosteric coupling

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Abstract

IIAGlc, the glucose-specific phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system, is an allosteric inhibitor of Escherichia coli glycerol kinase. A linked-functions initial-velocity enzyme kinetics approach is used to define the MgATP–IIAGlc heterotropic allosteric interaction. The interaction is measured by the allosteric coupling constants Q and W, which describe the mutual effect of the ligands on binding affinity and the effect of the allosteric ligand on Vmax, respectively. Allosteric interactions between these ligands display K-type activation and V-type inhibition. The allosteric coupling constant Q is about 3, showing cooperative coupling such that each ligand increases the affinity for binding of the other. The allosteric coupling constant W is about 0.1, showing that the allosteric inhibition is partial such that binding of IIAGlc at saturation does not reduce Vmax to zero. E. coli glycerol kinase is a member of the sugar kinase/heat shock protein 70/actin superfamily, and an element of the superfamily conserved ATPase catalytic core was identified as part of the IIAGlc inhibition network because it is required to transplant IIAGlc allosteric control into a non-allosteric glycerol kinase [A.C. Pawlyk, D.W. Pettigrew, Proc. Natl. Acad. Sci. USA 99 (2002) 11115–11120]. Two of the amino acids at this locus of E. coli glycerol kinase are replaced with those from the non-allosteric enzyme to enable determination of its contributions to MgATP–IIAGlc allosteric coupling. The substitutions reduce the affinity for IIAGlc by about 5-fold without changing significantly the allosteric coupling constants Q and W. The insensitivity of the allosteric coupling constants to the substitutions may indicate that the allosteric network is robust or the locus is not an element of that network. These possibilities may arise from differences of E. coli glycerol kinase relative to other superfamily members with respect to oligomeric structure and location of the allosteric site in a single domain far from the catalytic site.

Section snippets

Materials

Reagents were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise indicated. IIAGlc was prepared as described [45] from the BL21(DE3) strain of E. coli bearing the pVEX-crr plasmid which was generously provided by Drs. Norman Meadow and Saul Roseman of the Department of Biology of The Johns Hopkins University, Baltimore, MD. The concentration of IIAGlc was determined from absorbance at 260 nm by using an extinction coefficient of 1.6 mM−1 cm−1, which was determined by using

Results

The enzymes that are used here have the substitution E478C in the IIAGlc-binding site, which is denoted by the letter C in the enzyme name. The E478C substitution increases the affinity for IIAGlc, which facilitates these determinations of the allosteric coupling. The concentration of IIAGlc that gives half-maximal inhibition of EGK is about 10 μM, which is decreased to about 1 μM by the addition of ZnCl2 or by the amino acid substitution E478C [47]. The apparent affinity is increased by Zn(II)

Discussion

The objective of these studies is determination of the role of the coupling locus in the allosteric network that couples the allosteric and catalytic sites, for which measurement of the allosteric coupling parameters is required. The allosteric parameters that describe the coupling between binding of IIAGlc and MgATP and the effect of IIAGlc on Vmax for EGKC and EGKC-VN are determined by using a linked-functions initial-velocity enzyme kinetics approach. Results from this approach show that

Acknowledgments

This work was supported by Grant GM068768 from the National Institutes of Health and by Texas AgriLife Research (formerly Texas Agricultural Experiment Station). The author thanks Jesse Flynn, Rebecca Jurrens, Pamela S. Miller, Brandon Sexton, and Jillian Wisdom for expert technical assistance; the Protein Chemistry Laboratory of Texas A&M University for performing amino acid analysis for determination of the extinction coefficient for IIAGlc; the Gene Technologies Laboratory of Texas A&M

References (60)

  • J. Monod et al.

    J. Mol. Biol.

    (1965)
  • E.J. Fuentes et al.

    J. Mol. Biol.

    (2006)
  • Z. Li et al.

    J. Mol. Biol.

    (2006)
  • Z. Shi et al.

    Curr. Opin. Struct. Biol.

    (2006)
  • W. Rist et al.

    J. Biol. Chem.

    (2006)
  • D. Grueninger et al.

    J. Mol. Biol.

    (2006)
  • A.E. Aleshin et al.

    J. Mol. Biol.

    (2000)
  • P. Graceffa et al.

    J. Biol. Chem.

    (2003)
  • R. Page et al.

    J. Mol. Biol.

    (1998)
  • K. Kamata et al.

    Structure

    (2004)
  • R. Dominguez

    TIBS

    (2004)
  • Q. Liu et al.

    Cell

    (2007)
  • M.D. Feese et al.

    Structure

    (1998)
  • C. Frieden

    J. Biol. Chem.

    (1964)
  • G.D. Reinhart
  • G.D. Reinhart

    Arch. Biochem. Biophys.

    (1983)
  • M.M. Symcox et al.

    Anal. Biochem.

    (1992)
  • B.L. Braxton et al.

    J. Biol. Chem.

    (1994)
  • V.L. Tlapak-Simmons et al.

    Arch. Biochem. Biophys.

    (1994)
  • F. Rosseau et al.

    Curr. Opin. Struct. Biol.

    (2005)
  • M.D. Daily et al.

    Proteins

    (2008)
  • T. Liu et al.

    Proc. Natl. Acad. Sci. USA

    (2007)
  • M.W. Clarkson et al.

    Biochemistry

    (2006)
  • N. Goodey et al.

    Nat. Chem. Biol.

    (2008)
  • G. Suel et al.

    Nat. Struct. Biol.

    (2003)
  • G.K. Ackers et al.

    Annu. Rev. Biochem.

    (1985)
  • A. del Sol et al.

    Genome Biol.

    (2007)
  • R. Dima et al.

    Protein Sci.

    (2006)
  • M.D. Daily et al.

    Proteins

    (2007)
  • R. Amaro et al.

    Biochemistry

    (2007)
  • Cited by (0)

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