Mechanistic studies of the inactivation of low molecular weight protein tyrosine phosphatases
Abstract
This thesis examines various inhibitors of the low molecular weight protein tyrosine phosphatases (PTPases). Phenylarsine oxide (PAO) is a widely used PTPase-specific inhibitor. PAO is known for its interaction of vicinal dithiols in proteins. However, only the low Mr PTPases possess vicinal dithiols at the enzyme active site. The results of this study suggest that PAO inhibits various classes of PTPases via different mechanisms. Naphthoquinone derivatives (NQs) have been previously studied for their potential roles as anti-cancer therapeutic agents. It has been speculated that one possible reaction in the cell might be a NQ-induced inhibition and inactivation of protein tyrosine phosphatases (PTPases) by reaction with an active site cysteine residue. The present study demonstrates the inactivation of several low molecular weight protein tyrosine phosphatases by naphthoquinone (NQ) derivatives and examines the detailed mechanism of the process. The inactivation of low Mr PTPases by 1,2-naphthoquinone (1,2NQ) derivatives was found to proceed through at least two steps: a fast equilibrium binding at the enzyme active site, with 1,2NQ acting as a competitive inhibitor, followed by a slower covalent arylation of active site cysteine(s). When 1,2NQ acted as a competitive inhibitor of a human low molecular weight PTPase isoenzyme (HPTPA), the Ki was found to be 7 μM at pH 7.0. It was also determined that in the presence of air, NQ could participate in redox reactions leading to the formation of reactive oxygen species and the subsequent oxidization of the two active site cysteines (unique to low molecular weight PTPases), which in turn led to the formation of a disulfide bond and a resulting inactivation of the enzyme. Simple thiols, such as glutathione at cellular concentrations, did not significantly retard the reaction of NQs with PTPases. Based on these results, it is likely that NQ derivatives can both directly and indirectly inactivate PTPases in the cell.
Degree
Ph.D.
Advisors
Etten, Purdue University.
Subject Area
Biochemistry|Analytical chemistry
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