FUNCTIONAL-EVALUATION OF INVARIANT ARGININES SITUATED IN THE MOBILE LID DOMAIN OF PHOSPHORIBULOKINASE

Rhodobacter sphaeroides phosphoribulokinase contains four invariant ar ginines (R49, R168, R173, and R187). The high-resolution structure of this enzyme [Harrison, D. H. T., Runquist, J. A., Holub, A., and Mizio rko, H. M. (1998) Biochemistry (submitted for publication)] reveals th at it folds in a manner similar to that of adenylate kinase. Three inv ariant arginines (R168, R173, and R187) as well as arginine-186, which is conserved in prokaryotic phosphoribulokinases, have not been previ ously functionally evaluated. These arginine residues map within the m obile lid domain that is a distinctive feature of the adenylate kinase family of proteins. Precedent for the significant function of arginin es in phosphotransferase reactions prompted substitution of glutamine for each of these three invariant arginines. Solution state characteri zation of the isolated mutant proteins indicated that they retained a high degree of structural integrity, as indicated by their stoichiomet ric binding of an alternative nucleotide substrate (trinitrophenyl-ATP ) as well as the allosteric effector (NADH). Kinetic characterization indicated > 10(4)-fold diminution in V/K-Ru5P for R168Q, attributable to a >300-fold decrease in catalytic efficiency and an increase (simil ar to 50-fold) in K-m Ru5P. For R173Q, a 15-fold diminution in V-max a nd a 100-fold increase in K-m Ru5P were observed. These observations i mplicate new components of the ribulose 5-phosphate binding site. Addi tionally, they confirm assignment of the mobile lid domain as part of the phosphoribulokinase active site, even though this region is well s eparated from other active site elements in the structure of the open form of the protein. Characterization of R186Q and R187Q mutants sugge sts that they influence the cooperativity of substrate binding.