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

Abstract

IIA Glc , the glucose-specific phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system, is an allosteric inhibitor of Escherichia coli glycerol kinase. A linkedfunctions initial-velocity enzyme kinetics approach is used to define the MgATP-IIA Glc 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 V max , respectively. Allosteric interactions between these ligands display K-type activation and Vtype 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 IIA Glc at saturation does not reduce V max 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 IIA Glc inhibition network because it is required to transplant IIA Glc allosteric control into a non-allosteric glycerol kinase (Pawlyk, A.C. and Pettigrew, D.W. (2002) Proc. Natl. Acad. Sci. USA 99:11115-20). 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-IIA Glc allosteric coupling. The substitutions reduce the affinity for IIA Glc 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.Allosteric control of enzyme catalysis is a central aspect of regulation in biological systems. In central metabolic pathways, modulation of catalytic activity coordinates responses to changing carbon source availability, energy requirements, or needs for specific molecules for anabolism. Classical descriptions of allostery are based on global conformational change in response to binding of a regulatory ligand to its allosteric site that results in change in the conformation and function of the catalytic site [1]. Developing views of allostery differ in several respects from the classical description. Among the most important of these differences *To whom correspondence should be addressed to Texas A&M University, Department of Biochemistry & Biophysics, 2128 TAMU, College Station, TX 77843-2128. email address: dpettigrew@tamu.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our...

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