Modification of the Active Site of Alkaline Phosphatase by Site-Directed Mutagenesis
- 10 January 1986
- journal article
- research article
- Published by American Association for the Advancement of Science (AAAS) in Science
- Vol. 231 (4734) , 145-148
- https://doi.org/10.1126/science.3510454
Abstract
The catalytically essential amino acid in the active site of bacterial alkaline phosphatase (Ser-102) has been replaced with a cysteine by site-directed mutagenesis. The resulting thiol enzyme catalyzes the hydrolysis of a variety of phosphate monoesters. The rate-determining step of hydrolysis, however, is no longer the same for catalysis when the active protein nucleophile is changed from the hydroxyl of serine to the thiol of cysteine. Unlike the steady-state kinetics of native alkaline phosphatase, those of the mutant show sensitivity to the leaving group of the phosphate ester.Keywords
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