Cobalt activation of Escherichia coli 5'-nucleotidase is due to zinc ion displacement at only one of two metal-ion-binding sites.
Escherichia coli 5'-nucleotidase activity is stimulated 30- to 50-fold in vitro by the addition of Co(2+). Seven residues from conserved sequence motifs implicated in the catalytic and metal-ion-binding sites of E. coli 5'-nucleotidase (Asp(41), His(43), Asp(84), His(117), Glu(118), His(217) and His(252)) were selected for modification using site-directed mutagenesis of the cloned ushA gene. On the basis of comparative studies between the resultant mutant proteins and the wild-type enzyme, a model is proposed for E. coli 5'-nucleotidase in which a Co(2+) ion may displace the Zn(2+) ion at only one of two metal-ion-binding sites; the other metal-ion-binding site retains the Zn(2+) ion already present. The studies reported herein suggest that displacement occurs at the metal-ion-binding site consisting of residues Asp(84), Asn(116), His(217) and His(252), leading to the observed increase in 5'-nucleotidase activity.
McMillen
Lyle
L
School of Biomolecular and Biomedical Science, Faculty of Science, Griffith University, Nathan, Brisbane, Queensland 4111, Australia.
Beacham
Ifor R
IR
Burns
Dennis M
DM
D015720:5'-Nucleotidase / Q000378:metabolism*; D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D003035:Cobalt / Q000494:pharmacology*; D017931:DNA Primers / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008670:Metals / Q000032:analysis / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010727:Phosphoric Diester Hydrolases / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism*
D017931:DNA Primers; D029968:Escherichia coli Proteins; D008670:Metals; C406334:ushA protein, E coli; D003035:Cobalt; D015720:5'-Nucleotidase; D010727:Phosphoric Diester Hydrolases; D015032:Zinc