Alkaline phosphatase (AP) is a ubiquitous enzyme responsible for maintaining inorganic phosphate concentrations and providing hydrolysis of a variety of substrates across the AP superfamily. Changes within the active site often yield decreased catalytic efficiency due to the importance of positioning active site metals. Interestingly, alterations in the entrance to the active site are found across species with an extreme evolution of position 100 from threonine to the structurally restrictive proline residue. Given the increased activity of mammalian AP relative to <i>E. coli</i> and the close proximity of this residue to the active site serine 102, this single amino acid change was mimicked in <i>E. coli</i> to assess the structural and mechanistic impact of this proline evolution. Initial studies indicate a significant increase in catalytic efficiency despite a reduction in binding affinity as well as a reduction in the global stability of the enzyme structure. Further pH dependent studies will elucidate the impact of this mutation in the catalytic mechanism of this promiscuous enzyme.

Kinky evolution of the alkaline phosphatase active site yields increased catalytic efficiency and promiscuity

2019 Spring National Meeting

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