Investigation of Effects WPD/IPD-loop Mutations in Specific Protein Tyrosine Phosphatases

Phosphorylation is an important mechanism that cells utilize to activate or deactivate receptors, regulators, and enzymes. A few protein tyrosine phosphatases (PTPs) were studied in this project; one of which is PTP1B, a down-regulator for the human insulin receptor. Another enzyme studied is YopH, a virulence agent in Yersinia pestis, the bacteria responsible for the bubonic plague. YopH is the fastest phosphatase with over 1,000 turnovers per second. The last enzyme focused on in this project is SSo, a PTP found in the thermophilic bacteria Sulfolobus solfataricus. Although the active site of each of these enzymes is highly superimposable, their rates of reaction are dramatically different. A mobile protein loop called the WPD loop at the active site in PTP1B and YopH is thought to have a large impact upon their reaction rates. This project has measured the effects of the mutation of tryptophan (W) of the WPD loop in PTP1B and YopH to the Isoleucine (I) present in the corresponding position in the SSo IPD loop, and the mutation of I to W in the Sso enzyme. Successful mutagenesis has been confirmed for PTP1B and YopH. Rates of reaction have been reduced in both enzymes, as hypothesized to result from the change of the amino acid side chain from a planar aromatic group to a branched hydrocarbon. However, the catalytic rate of the YopH mutant was reduced much more dramatically than the PTP1B mutant, by three orders of magnitude for YopH as opposed to one order of magnitude for PTP1B. The effects of the I to W mutation on SSo catalysis have not yet been assessed