Oligomeric interactions provide alternatives to direct steric modes of control of sugar kinase/actin/hsp70 superfamily functions by heterotropic allosteric effectors: inhibition of E. coli glycerol kinase.
Unlike those for monomeric superfamily members, heterotropic allosteric effectors of the tetrameric Escherichia coli glycerol kinase (EGK) bind to only one of the two domains that define the catalytic cleft and far from the active site. An R369A amino acid substitution removes oligomeric interactions of a novel mini domain-swap loop of one subunit with the catalytic site of another subunit, and an A65T substitution perturbs oligomeric interactions in a second interface. Linked-functions enzyme kinetics, analytical ultracentrifugation, and FRET are used to assess effects of these substitutions on the allosteric control of catalysis. Inhibition by phosphotransferase system protein IIA(Glc) is reduced by the R369A substitution, and inhibition by fructose 1,6-bisphosphate is abolished by the A65T substitution. The oligomeric interactions enable the heterotropic allosteric effectors to act on both domains and modulate the catalytic cleft closure despite binding to only one domain.
Pettigrew
Donald W
DW
Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128, USA. dpettigrew@tamu.edu
D000199:Actins / Q000737:chemistry*; D000409:Alanine / Q000235:genetics; D000494:Allosteric Regulation / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D019281:Dimerization; D004347:Drug Interactions; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005991:Glycerol Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005810:Multigene Family; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism
D000199:Actins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D021122:Protein Subunits; D001120:Arginine; D005991:Glycerol Kinase; D000409:Alanine