Escherichia coli glycerol kinase: evaluation of the roles of residue arginine 479 in the interaction with the phosphotransferase system regulatory protein IIIglc

Abstract

Protein-protein interactions are important for many biological processes and attract much interest these days. Electrostatic interactions are among the major forces determining the strength of protein-protein interactions. The complex formed by glycerol kinase (GK) and its protein inhibitor Factor lllglc provides a good model to study the importance of salt bridge in protein-protein interactions. In E. coli, the catalytic activity of GK is inhibited by Factor IIIglc through protein-protein interactions. The crystal structure of the complex of GK with dephospho-lllglc shows that residue arginine 479 (R479) of GK forms a buried salt bridge with residue aspartate 38 (D38) of lllglc. In a modeled complex of HPr-P-HAgIc (Illglc homologue in Bacillus subtilis), an arginine residue in HPr forms two salt bridges with an aspartate residue in the same region occupied by D38 in lllglc. Changing the arginine 479 to aspartate (R479D) abolishes III91C inhibition. These results suggest that residue R479 is crucial for formation of the complex between GK and Factor lllglc. Two mutations in GK, R479M and R479K, have been constructed by site-directed mutagenesis to investigate the role of residue R479 in salt bridge formation. The work reported here includes purification and characterization of the catalytic and regulatory properties of these mutant proteins. Both proteins can be purified like the wild type GK. The mutant proteins have little effect on the kinetic properties of the GK and its inhibition by FBP. Both mutations affect inhibition by Factor lllglc. The R479M mutation increases the dissociation constant (Ki) 10 fold, corresponding to a difference in free energy change of 1.7 Kcal/mol. The R479K mutation increases the Ki 5 fold, corresponding to a difference in free energy change of 0.8 Kcal/mol. Ammonium ion decreases the Ki value of Hlglc regulation for the R479M mutation slightly, but has essentially no effect on the wild type GK. Based on our results, the intermolecular salt bridge contributes only 1.7 Kcal/mol to the stability of the complex.