Functional evaluation of invariant arginines situated in the mobile lid domain of phosphoribulokinase.

摘要: Rhodobacter sphaeroides phosphoribulokinase contains four invariant arginines (R49, R168, R173, and R187). The high-resolution structure of this enzyme [Harrison, D. H. T., Runquist, J. A., Holub, Miziorko, M. (1998) Biochemistry (submitted for publication)] reveals that it folds in a manner similar to adenylate kinase. Three (R168, R187) as well arginine-186, which is conserved prokaryotic phosphoribulokinases, have not been previously functionally evaluated. These arginine residues map within the mobile lid domain distinctive feature kinase family proteins. Precedent significant function phosphotransferase reactions prompted substitution glutamine each these three arginines. Solution state characterization isolated mutant proteins indicated they retained high degree structural integrity, by their stoichiometric binding an alternative nucleotide substrate (trinitrophenyl-ATP) allosteric effector (NADH). Kinetic > 10(4)-fold diminution V/KRu5P R168Q, attributable 300-fold decrease catalytic efficiency increase (approximately 50-fold) Km Ru5P. For R173Q, 15-fold Vmax 100-fold Ru5P were observed. observations implicate new components ribulose 5-phosphate site. Additionally, confirm assignment part active site, even though region separated from other site elements open form protein. Characterization R186Q R187Q mutants suggests influence cooperativity binding.