<i>Escherichia coli</i> alkaline phosphatase (AP) contains a complex active site responsible for the hydrolysis of phosphate from a variety of substrates and for cross superfamily sulfatase activity.  A hydrophobic platform located below the AP active site is thought to play a key role in maintaining the structure of the active site and allowing for enzymatic activity. To study the role of this platform in structural integrity, catalytic activity and promiscuity of this enzyme a more significant hydrophobic aromatic group was introduced through a Y84W, single amino acid mutation. Alterations to this platform yield a significant increase in catalytic efficiency despite a decrease in binding affinity. A novel fluorescence assay, used to determine the relative structural stability of the Y84W mutant relative to WT, indicates global destabilization of the AP active site creating facile release of product and hence increased catalytic activity. Further studies into the mechanistic changes due to alteration in the hydrophobic platform via pre-steady state kinetics will further elucidate the role of this platform in modulating structural stability and catalytic activity.

Altering a hydrophobic platform in the <i>Escherichia coli</i> alkaline phosphatase active site

2019 Spring National Meeting

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Altering a hydrophobic platform in the Escherichia coli alkaline phosphatase active site