title: Functional evaluation of serine/threonine residues in the P-Loop of Rhodobacter sphaeroides phosphoribulokinase.
abstract: The N-terminal region of phosphoribulokinase (PRK) has been proposed to contain a "P-loop" or "Walker A" motif. In Rhodobacter sphaeroides PRK, 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 x 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 PRKMg-ATP complexes. Each mutant complex is characterized by (31)P resonances for alpha- and gamma-phosphoryls of bound Mg-ATP which are narrower than measured for wild-type PRKMg-ATP; signals for the beta-phosphoryl are poorly detectable for mutant PRKMg-ATP complexes. Kinetic characterization indicates that these mutants differ markedly with respect to catalytic activity. T20A exhibits V(m) comparable to wild-type PRK, while V(m) is diminished by 8-fold for T18A and by 40-fold for S14A. In contrast to these modest effects, S19A exhibits decreases in V(m) and V(m)/K(Ru5P) of 500-fold and >15000-fold, respectively. S19A and T18A exhibit only modest (6-7-fold) increases in S(1/2) for ATP but larger (30-45-fold) increases in K(m) for Ru5P. K(I) 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.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Runquist
authors.forename: S E
authors.initials: SE
authors.lastname: Ríos
authors.forename: D A
authors.initials: DA
authors.lastname: Vinarov
authors.forename: H M
authors.initials: HM
authors.lastname: Miziorko
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*
chemical_list: D008024:Ligands; D010446:Peptide Fragments; D009243:NAD; D013912:Threonine; D012694:Serine; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase