Functional Evaluation of Serine/Threonine Residues in the P-Loop of Rhodobacter sphaeroides Phosphoribulokinase †

The N-terminal region of phosphoribulokinase (PRK) has been proposed to contain a "P- loop" or "Walker A" motif. In Rhodobacter sphaeroidesPRK, four alcohol side chains, contributed by S14, T18, S19, and T20, map within the P loop and represent potential Mg-ATP ligands. Each of these has been individually replaced with an alanine and the impact of these substitutions on enzyme-ATP interactions and overall catalytic efficiency evaluated. Each mutant PRK retains the ability to tightly bind the positive effector, NADH (0.7-0.9 per site), and exhibits allosteric activation, suggesting that the proteins retain a high degree of structural integrity. Similarly, each mutant PRK retains the ability to stoichiometrically (0.7-1.2 per site) bind the alternative substrate trinitrophenyl-ATP. Despite the large size of the PRK oligomer (8 32 kDa), 31 P NMR can be used to detect stoichiometrically bound Mg- ATP substrate, which produces markedly broadened peaks in comparison with signals from unbound Mg-ATP. Elimination of alcohol substituents in mutants T18A, S19A, or T20A produces enzymes which retain the ability to form stable PRK/Mg-ATP complexes. Each mutant complex is characterized by 31 P resonances for R- and A-phosphoryls of bound Mg-ATP which are narrower than measured for wild- type PRK/Mg-ATP; signals for the ‚-phosphoryl are poorly detectable for mutant PRK/Mg-ATP complexes. Kinetic characterization indicates that these mutants differ markedly with respect to catalytic activity. T20A exhibits Vm comparable to wild-type PRK, while Vm is diminished by 8-fold for T18A and by 40-fold for S14A. In contrast to these modest effects, S19A exhibits decreases in Vm and Vm/KRu5P of 500-fold and >15000-fold, respectively. S19A and T18A exhibit only modest (6-7-fold) increases in S1/2 for ATP but larger (30-45-fold) increases in Km for Ru5P. KI values for the competitive inhibitor, 6-phosphogluconate, do not significantly change upon mutation of T18 or S19, suggesting that these residues are not crucial to Ru5P binding. A role for the alcohol group of S19, the eighth residue in P-loop motif, as a ligand to the Mg-ATP substrate seems compatible with the characterization data; adjacent alcohols do not efficiently function as surrogates. Such a proposed function for S19 is compatible with its proximity to E131, the acidic residue in a putative Walker B motif and probable second Mg-ATP ligand in PRK's active site.