Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat

• Published in: Protein engineering 1991-10, Vol.4 (7), p.801-804
• Main Authors: Mandecki, Wlodek, Shallcross, Mary Ann, Sowadski, Janusz, Tomazic-Allen, Susan
• Format: Article
• Language: English
• Subjects: Alkaline Phosphatase - genetics; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Binding Sites - genetics; Biological and medical sciences; calalysis; DNA Mutational Analysis; Enzymes and enzyme inhibitors; Escherichia coli - genetics; evolution; Fundamental and applied biological sciences. Psychology; Gene Expression; Hydrogen-Ion Concentration; Hydrolases; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; sequence homology; Sequence Homology, Nucleic Acid; specific activity; Structure-Activity Relationship
• ISSN: 1741-0126; 0269-2139; 1741-0134; 1460-213X
• DOI: 10.1093/protein/4.7.801

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 tra,. Of –170 mutant enzymes screened, 5% (eight mutants) exhibited significant increases in specific activity. In particular, a substitution by serine of a totally invariant AsplOl resulted in a 35-fold increase of specific activity over wild-type at pH 10.0. Up to 6-fold increases the kcat/km ratio were observed.