Evidence supporting catalytic roles for aspartate residues in phosphoribulokinase.
The DNA encoding Rhodobacter sphaeroides phosphoribulokinase (PRK) has been modified to allow ligation into pET-3d. Using the resulting expression plasmid, PRK was overexpressed in Escherichia coli and isolated in milligram quantities. Homogeneous preparations of the enzyme exhibit properties comparable to those of PRK expressed using a previously described pUC19-derived construct [Sandbaken et al., Biochemistry 31, 3715-3719]. Mutagenesis experiments have been designed to produce conservative substitutions that eliminate the carboxyl groups of each of four conserved acidic residues (D42, E131, D169, and E178). Using the newly developed expression system, the resulting PRK variants have been expressed, isolated, and characterized. Expression levels and recoveries upon affinity chromatography purification are similar to the results obtained with wild-type PRK. Apparent substrate affinities of these mutant proteins do not differ greatly from values observed for wild-type PRK. In contrast, these PRK variants display a wide range of Vmax values, ranging from wild-type activity (approximately 200 units/mg; E178A) to levels that are diminished by 4 (D169A) to 5 (D42A, D42N) orders of magnitude. That the large diminutions in catalytic activity are significant and do not merely reflect gross perturbations in protein structure is suggested not only by the modest effects on substrate affinity but also by the allosteric properties of D169A, D42A, and D42N. The activities of these proteins, like that of wild-type PRK, are markedly stimulated by the positive effector NADH. The magnitude of the Vmax perturbations suggests that D42 and D169 are candidates for the role of active site base or activator cation ligand.(ABSTRACT TRUNCATED AT 250 WORDS)
Charlier
H A
HA
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Runquist
J A
JA
Miziorko
H M
HM
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D005798:Genes, Bacterial; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D005971:Glutamates; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase