Identification of the phosphoribulokinase sugar phosphate binding domain.
A recombinant form of Rhodobacter sphaeroides phosphoribulokinase (form I; NADH dependent) has been expressed in and purified to homogeneity from Escherichia coli that harbor the prkA gene in the plasmid pKP1565b. Restriction digestion of the phosphoribulokinase-encoding plasmid produces a tractable 450 bp fragment that encodes amino acid residues 28-179, which include a region (residues 42-54) highly conserved among phosphoribulokinase proteins. Using overlap extension polymerase chain reaction methodology, directed mutagenesis was performed to produce mutant proteins in which basic residues in this conserved region were replaced by neutral amino acids. Lysine-53, implicated by affinity labeling studies, has been replaced by methionine; little effect on substrate binding or catalysis is apparent. In contrast, when histidine-45 is replaced by asparagine, a 40-fold increase in the Km for ribulose 5-phosphate results; a 200-fold increase results when arginine-49 is replaced by glutamine. Implication of this region as part of the sugar phosphate binding site is compatible with previous results that indicate targeting by an ATP analogue containing a reactive functionality esterified to the gamma-phosphoryl group. The phosphoribulokinase reaction involves a single in-line phosphoryl transfer, requiring that the gamma-phosphoryl of ATP be closely juxtaposed to the bound cosubstrate. It follows that any reactive group attached to the gamma-phosphoryl in a nucleotide analogue that is bound to PRK in the absence of the cosubstrate will be favorably positioned to modify the sugar phosphate binding site.
Sandbaken
M G
MG
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Runquist
J A
JA
Barbieri
J T
JT
Miziorko
H M
HM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D012689:Sequence Homology, Nucleic Acid; D013403:Sugar Phosphates / Q000378:metabolism*
D011994:Recombinant Proteins; D013403:Sugar Phosphates; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase