Ja. Runquist et al., FUNCTIONAL-EVALUATION OF INVARIANT ARGININES SITUATED IN THE MOBILE LID DOMAIN OF PHOSPHORIBULOKINASE, Biochemistry, 37(5), 1998, pp. 1221-1226
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.