Escherichia coli 5′-nucleotidase activity is stimulated 30- to 50-fold in vitro by the addition of Co2+. Seven residues from conserved sequence motifs implicated in the catalytic and metal-ion-binding sites of E. coli 5′-nucleotidase (Asp41, His43, Asp84, His117, Glu118, His217 and His252) 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 Co2+ ion may displace the Zn2+ ion at only one of two metal-ion-binding sites; the other metal-ion-binding site retains the Zn2+ ion already present. The studies reported herein suggest that displacement occurs at the metal-ion-binding site consisting of residues Asp84, Asn116, His217 and His252, leading to the observed increase in 5′-nucleotidase activity.
P-C5-16 Helix packing in the lactose permease of Escherichia coli as determined by use of designed metal ion binding sites and site-directed spin labeling