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
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
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