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The inactivation mechanism of low molecular weight phosphotyrosine-protein phosphatase by H2O2
A Caselli1, R Marzocchini, G Camici
1Dipartimento di Scienze Biochimiche, Università di Firenze, Viale Morgagni 50, 50134 Florence, Italy.
Abstract:
Low molecular weight phosphotyrosine-protein phosphatase (LMW-PTP) shares no general sequence homology with other PTPs, although it has an active site sequence motif CXXXXXR and a reaction mechanism identical to those of all PTPs. The main function of this enzyme is the down-regulation of platelet-derived growth factor and insulin receptors. Both human LMW-PTP isoenzymes are inactivated by H2O2. The enzymes are protected from inactivation by Pi, a competitive inhibitor, suggesting that the H2O2 reaction is directed to active site. Analysis of free thiols performed on the inactivated enzymes demonstrates that only two out of the eight LMW-PTP cysteines are modified. Time-course high performance liquid chromatography-electrospray mass spectrometry, together with specific radiolabeling and tryptic fingerprint analyses, enables us to demonstrate that H2O2 causes the oxidation of Cys-12 and Cys-17 to form a disulfide bond. Because both residues are localized into the active site region, this modification inactivates the enzyme. Fluorescence spectroscopy experiments suggest that the fold of the enzyme is modified during oxidation by H2O2. Because a physiological concentration of H2O2 produces enzyme inactivation and considering that the activity is restored by reduction with low molecular weight thiols, we suggest that oxidative stress conditions and other processes producing hydrogen peroxide regulate the LMW-PTP in the cell.
Insights
Low molecular weight phosphotyrosine-protein phosphatase (LMW-PTP) is inactivated by hydrogen peroxide (H2O2) through disulfide bond formation at its active site. This oxidative stress mechanism regulates LMW-PTP activity in cells.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Low molecular weight phosphotyrosine-protein phosphatase (LMW-PTP) regulates key growth factor and insulin receptors.
- LMW-PTP lacks homology with other protein tyrosine phosphatases (PTPs) but shares a conserved active site motif (CXXXXXR) and catalytic mechanism.
Purpose of the Study:
- To elucidate the mechanism by which hydrogen peroxide (H2O2) inactivates LMW-PTP.
- To identify the specific residues involved in H2O2-mediated inactivation and understand its regulatory implications.
Main Methods:
- High-performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI-MS) for time-course analysis.
- Radiolabeling and tryptic fingerprinting to identify modified residues.
- Fluorescence spectroscopy to assess enzyme structural changes.
Main Results:
- H2O2 inactivates LMW-PTP by forming a disulfide bond between Cys-12 and Cys-17, located in the active site.
- Only two of the eight cysteine residues in LMW-PTP are modified by H2O2.
- Oxidation alters the enzyme's fold, leading to inactivation, which can be reversed by reducing agents.
Conclusions:
- Hydrogen peroxide inactivates LMW-PTP via active site cysteine oxidation and disulfide bond formation.
- Cellular regulation of LMW-PTP activity may occur through oxidative stress and hydrogen peroxide signaling.
- Understanding this mechanism is crucial for comprehending cellular signaling pathways involving growth factors and insulin.