Mutagenesis of conserved residues within the active site of Escherichia coli alkaline phosphatase yields enzymes with increased kcat |
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Authors: | Mandecki Wlodek; Shallcross Mary Ann; Sowadski Janusz; Tomazic-Allen Susan |
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Affiliation: | Corporate Molecular Biology San Diego, USA
2Department of Biology, University of California San Diego, USA
3Probe Diagnostics Venture, Abbott Laboratories Abbott Park, IL 60064 |
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Abstract: | The likelihood for improvement in the catalytic properties ofEscherichia coli alkaline phosphatase was examined using site-directedmutagenesis. Mutants were constructed by introducing sequencechanges into nine preselected amino acid sites within 10 A ofthe catalytic residue serine 102. When highly conserved residuesin the family of alkaline phosphatases were mutated, many ofthe resulting enzymes not only maintained activity, but alsoexhibited greatly improved tra,. Of 170 mutant enzymesscreened, 5% (eight mutants) exhibited significant increasesin specific activity. In particular, a substitution by serineof a totally invariant AsplOl resulted in a 35-fold increaseof specific activity over wild-type at pH 10.0. Up to 6-foldincreases the kcat/km ratio were observed. |
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Keywords: | calalysis/ evolution/ gene expression/ sequence homology/ specific activity |
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