Transplanting allosteric control of enzyme activity by protein-protein interactions: coupling a regulatory site to the conserved catalytic core

Abstract

Glycerol kinase from Escherichia coli, but not Haemophilus influenzae, is inhibited allosterically by phosphotransferase system protein IIA(Glc). The primary structures of these related kinases contain 501 amino acids, differing at 117. IIA(Glc) inhibition is transplanted from E. coli glycerol kinase into H. influenzae glycerol kinase by interconverting only 11 of the differences: 8 residues that interact with IIA(Glc) at the allosteric binding site and 3 residues in the conserved ATPase catalytic core that do not interact with IIA(Glc) but the solvent accessible surface of which decreases when it binds. The three core residues are crucial for coupling the allosteric site to the conserved catalytic core of the enzyme. The site of the coupling residues identifies a regulatory locus in the sugar kinase/heat shock protein 70/actin superfamily and suggests relations between allosteric regulation and the active site closure that characterizes the family. The location of the coupling residues provides empirical validation of a computational model that predicts a coupling pathway between the IIA(Glc)-binding site and the active site [Luque, I. & Freire, E. (2000) Proteins Struct. Funct. Genet. Suppl. 4, 63-71]. The requirement for changes in core residues to couple the allosteric and active sites and switching from inhibition to activation by a single amino acid change are consistent with a postulated mechanism for molecular evolution of allosteric regulation.

Fig 1.

Ribbon diagrams of the structure of one EcGK subunit with bound IIA^Glc. IIA^Glc is shown in magenta, and EcGK is shown in the remaining colors. (A) The conserved ATPase catalytic core. Glycerol is shown in green in the active site. Domain II, which contains the IIA^Glc binding site, is to the left of the cleft, and domain I is to the right. The conserved ATPase motif in domain I is shown in red and in domain II in gray. The secondary structure elements α₃ and β₅ are labeled in each domain. (B) Amino acid differences in EcGK and HiGK. Identities are shown in blue, conservative substitutions in red, and nonconservative substitutions in gold. The N- and C-terminal positions are labeled 1 and 501, respectively, and selected residue positions are numbered. (C) The location of amino acid substitutions required to transplant IIA^Glc inhibition to HiGK. The amino acids that interact directly with IIA^Glc are shown as stick models in red. Amino acids 427–429 in EcGK (GTR) are shown in green and in HiGK (DVN) are shown in gold. D⁴²⁷ is labeled D and R⁴²⁹ is labeled R. Conserved ATPase catalytic core elements are colored as described for A. The loop with the binding site for FBP in domain I is shown in green and labeled FBP. The images were generated from PDB ID code by using SWISS-PDB VIEWER 3.5 (15) and rendered by using POV-RAY 3.1 for WINDOWS (www.povray.org/).

Fig 2.

Identification of EcGK primary structure that transplants IIA^Glc inhibition to HiGK. The primary structure of EcGK is represented by the thicker line, and that of HiGK is represented by the thinner line. Chimeric enzymes were constructed as described under Methods. IIA^Glc inhibition was assessed by determining the specific activity of glycerol kinase in cell extracts without and with the addition of IIA^Glc to the assay. +, IIA^Glc inhibits the chimeric enzyme; −, no IIA^Glc inhibition observed.