Mechanism of Inhibition of Protein-tyrosine Phosphatases by Vanadate and Pervanadate

doi:10.1074/jbc.272.2.843

Mechanism of Inhibition of Protein-tyrosine Phosphatases by Vanadate and Pervanadate*

From the ^‡ Department of Biochemistry and Molecular Biology and the
^∥ Department of Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research, 16711 Trans Canada Highway, Kirkland, Québec, H9H 3L1, Canada and the
^§ Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Blvd. West, Montréal, Québec, H3G 1M8, Canada

** To whom correspondence should be addressed:
Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe-Claire-Dorval, Québec, Canada H9R 4P8.
Tel.: 514-428-8543; Fax: 514-695-0693.

Abstract

Vanadate and pervanadate (the complexes of vanadate with hydrogen peroxide) are two commonly used general protein-tyrosine phosphatase (PTP) inhibitors. These compounds also have insulin-mimetic properties, an observation that has generated a great deal of interest and study. Since a careful kinetic study of the two inhibitors has been lacking, we sought to analyze their mechanisms of inhibition. Our results show that vanadate is a competitive inhibitor for the protein-tyrosine phosphatase PTP1B, with a K_i of 0.38 ± 0.02 μM. EDTA, which is known to chelate vanadate, causes an immediate and complete reversal of the inhibition due to vanadate when added to an enzyme assay. Pervanadate, by contrast, inhibits by irreversibly oxidizing the catalytic cysteine of PTP1B, as determined by mass spectrometry. Reducing agents such as dithiothreitol that are used in PTP assays to keep the catalytic cysteine reduced and active were found to convert pervanadate rapidly to vanadate. Under certain conditions, slow time-dependent inactivation by vanadate was observed; since catalase blocked this inactivation, it was ascribed to in situ generation of hydrogen peroxide and subsequent formation of pervanadate. Implications for the use of these compounds as inhibitors and rationalization for some of their in vivo effects are considered.

Footnotes

↵^¶ Supported by a Natural Sciences and Engineering Research Council of Canada 1967 Science and Engineering Studentship.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PTP

protein tyrosine phosphatase

DTT

dithiothreitol

FDP

fluorescein diphosphate

HPLC

high pressure liquid chromatography

MS

mass spectrometry

MS/MS

tandem mass spectrometry

capillary LC-MS

capillary HPLC-electrospray ionization mass spectrometry

VL

monoperoxovanadate

VL₂

diperoxovanadate.
↵²Huang, Z., Wang, Q., Ly, H., Govindarajan, A., Scheigetz, I., Zamboni, R., and Ramachandran, C., manuscript in preparation.
↵³A. Tracey, personal communication.
- Received April 2, 1996.
- Revision received October 16, 1996.