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.
IIA(Glc), 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-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 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 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 [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-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.
Pettigrew
Donald W
DW
Department of Biochemistry & Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128, USA. dpettigrew@tamu.edu
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005991:Glycerol Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000378:metabolism; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D007770:L-Lactate Dehydrogenase; D005991:Glycerol Kinase; D011770:Pyruvate Kinase; D000251:Adenosine Triphosphatases