Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat.
The likelihood for improvement in the catalytic properties of Escherichia coli alkaline phosphatase was examined using site-directed mutagenesis. Mutants were constructed by introducing sequence changes into nine preselected amino acid sites within 10 A of the catalytic residue serine 102. When highly conserved residues in the family of alkaline phosphatases were mutated, many of the resulting enzymes not only maintained activity, but also exhibited greatly improved kcat. Of approximately 170 mutant enzymes screened, 5% (eight mutants) exhibited significant increases in specific activity. In particular, a substitution by serine of a totally invariant Asp101 resulted in a 35-fold increase of specific activity over wild-type at pH 10.0. Up to 6-fold increases of the kcat/Km ratio were observed.
Mandecki
W
W
Corporate Molecular Biology, Abbott Laboratories, Abbott Park, IL 60064.
Shallcross
M A
MA
Sowadski
J
J
Tomazic-Allen
S
S
D000469:Alkaline Phosphatase / Q000235:genetics*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship
D000469:Alkaline Phosphatase