Functional contribution of a conserved, mobile loop histidine of phosphoribulokinase.
In the Rhodobacter sphaeroides phosphoribulokinase (PRK) structure, there are several disordered regions, including a loop containing invariant residues Y98 and H100. The functional importance of these residues has been unclear. PRK is inactivated by diethyl pyrocarbonate (DEPC) and protected by the substrates ATP and Ru5P, as well as by the competitive inhibitor, 6-phosphogluconate, suggesting active site histidine residue(s). PRK contains only three invariant histidines: H45, H100, and H134. Previous mutagenesis studies discount significant function for H134, but implicate H45 in Ru5P binding. PRK mutant H45N is inactivated by DEPC, implicating a second active site histidine. To evaluate the function of H100, as well as another invariant loop residue Y98, PRK mutants Y98L, H100A, H100N, and H100Q were characterized. Mutant PRK binding stoichiometries for the fluorescent alternative substrate, trinitrophenyl-ATP, as well as the allosteric activator, NADH, are comparable to wild-type PRK values, suggesting intact effector and substrate binding sites. The K(mRu5P) for the H100 mutants shows modest eight- to 14-fold inflation effects, whereas Y98L exhibits a 40-fold inflation for K(mRu5P). However, Y98L's K(i) for the competitive inhibitor 6-phosphogluconate is close to that of wild-type PRK. These observations suggest that Y98 and H100 are not essential Ru5P binding determinants. The Vm of Y98L is diminished 27-fold compared with wild-type PRK. In contrast, H100A, H100N, and H100Q exhibit significant decreases in Vm of 2600-, 2300-, and 735-fold, respectively. Results suggest that the mobile region containing Y98 and H100 must contribute to PRK's active site. Moreover, H100's imidazole significantly influences catalytic efficiency.
Runquist
Jennifer A
JA
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, 53226, USA.
Miziorko
Henry M
HM
D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004047:Diethyl Pyrocarbonate / Q000737:chemistry / Q000378:metabolism; D005069:Evaluation Studies as Topic; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017354:Point Mutation; D017433:Protein Structure, Secondary; D016415:Sequence Alignment
D006639:Histidine; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D004047:Diethyl Pyrocarbonate