Alkaline phosphatase is a ubiquitous, phosphomonoesterase that produces free inorganic phosphate required for cellular uptake and use. In addition to the E. coli alkaline phosphatase, there are four isozymes found in humans with variable activity and sequence homology to the bacterial phosphatase. The most significant sequence homology is found in the active site; however small variations in active site sequence do exist and are thought to be related to changes in structural stability, enzyme efficiency, and chemical mechanism. For example, in a variety of mammalian intestinal alkaline phosphatase, position 100 consists of a proline residue instead of the E. coli threonine. Interestingly, it has been demonstrated in past experiments that mammalian alkaline phosphatase has higher activity and a higher optimal pH for activity. These results are not surprising given the proximity of this residue to the active site nucleophile, serine 102. To test the effects of this evolved sequence change on both activity and structural stability, a single point mutation in E. coli alkaline phosphatase was made replacing threonine with proline (T100P). This mutant enzyme showed increased activity and, interestingly, a decrease in stability relative to wild type. These results suggest that the introduction of the hindered proline residue yields a change in active site architecture that results in improved release of product and a potential change in the rate-determining step for phosphomonoester cleavage.

Increasing the activity of E. coli alkaline phosphatase through a structurally destabilizing mutation

249th National Meeting (2015)

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