Altering of the metal specificity of Escherichia coli alkaline phosphatase.
Analysis of sequence alignments of alkaline phosphatases revealed a correlation between metal specificity and certain amino acid side chains in the active site that are metal-binding ligands. The Zn(2+)-requiring Escherichia coli alkaline phosphatase has an Asp at position 153 and a Lys at position 328. Co(2+)-requiring alkaline phosphatases from Thermotoga maritima and Bacillus subtilis have a His and a Trp at these positions, respectively. The mutations D153H, K328W, and D153H/K328W were induced in E. coli alkaline phosphatase to determine whether these residues dictate the metal dependence of the enzyme. The wild-type and D153H enzymes showed very little activity in the presence of Co(2+), but the K328W and especially the D153H/K328W enzymes effectively use Co(2+) for catalysis. Isothermal titration calorimetry experiments showed that in all cases except for the D153H/K328W enzyme, a possible conformation change occurs upon binding Co(2+). These data together indicate that the active site of the D153H/K328W enzyme has been altered significantly enough to allow the enzyme to utilize Co(2+) for catalysis. These studies suggest that the active site residues His and Trp at the E. coli enzyme positions 153 and 328, respectively, at least partially dictate the metal specificity of alkaline phosphatase.
Wojciechowski
Cheryl L
CL
Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Kantrowitz
Evan R
ER
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001419:Bacteria / Q000201:enzymology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002151:Calorimetry; D002413:Cations, Divalent / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D002413:Cations, Divalent; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase; D015032:Zinc; D002118:Calcium