Purification and kinetic characterization of the mitogen-activated protein kinase phosphatase rVH6.

The Journal of Biological Chemistry
A M WilandJ E Dixon

Abstract

The dual specificity protein-tyrosine phosphatase rVH6 belongs to a subfamily of enzymes that have in vivo and in vitro catalytic activity against mitogen-activated protein kinases. A method was developed for the expression and efficient purification of recombinant rVH6 in quantities sufficient for physical and kinetic characterization of the enzyme. Matrix-assisted laser desorption mass spectrometry verified the mass of purified rVH6 to be 43,500 +/- 150, and NH2-terminal sequence analysis confirmed the predicted amino acid sequence. Kinetic characterization of full-length rVH6 identified the critical ionizations involved in the kcat/Km parameter (apparent pKa values 5.1 and 6.6) and revealed a pH-independent kcat value of 0.014 s-1. In an attempt to define the essential catalytic core of this enzyme, amino acids 134-381 of rVH6 were expressed, purified, and characterized enzymatically. Kinetic analysis revealed that the truncated enzyme exhibited a turnover value similar to that of the full-length enzyme (kcat = 0.017 s-1), with p-nitrophenyl phosphate as substrate. Secondary structure prediction and molecular modeling of rVH6 based on the x-ray structure of the dual specificity protein tyrosine phosphatase, VHR, further supp...Continue Reading

References

Apr 27, 1979·Science·C M AndersonT A Steitz
May 1, 1992·Analytical Biochemistry·D J Hakes, J E Dixon
Mar 28, 1991·Nature·K L GuanJ E Dixon
Mar 1, 1991·Acta Crystallographica. Section A, Foundations of Crystallography·T A JonesM Kjeldgaard
Jun 20, 1995·Proceedings of the National Academy of Sciences of the United States of America·J M Denu, J E Dixon
Mar 16, 1995·Biochimica Et Biophysica Acta·S M Keyse
Jun 16, 1995·The Journal of Biological Chemistry·A Misra-PressP J Stork
Feb 24, 1995·The Journal of Biological Chemistry·J M DenuJ E Dixon
Mar 1, 1994·Proceedings of the National Academy of Sciences of the United States of America·Z Y ZhangJ E Dixon
Sep 1, 1995·Protein Science : a Publication of the Protein Society·H L SchubertM A Saper
Mar 19, 1996·Proceedings of the National Academy of Sciences of the United States of America·J M DenuJ E Dixon
May 31, 1996·Science·J YuvaniyamaM A Saper
Jun 1, 1996·Methods : a Companion to Methods in Enzymology·M Malmqvist

❮ Previous
Next ❯

Citations

Aug 23, 2002·Pharmacology & Therapeutics·Zhong-Yin ZhangLaiping Xie
Aug 3, 2011·Biochemistry·Victoria McParlandMaja Köhn
Apr 2, 2011·The Journal of Biological Chemistry·Yuan-Yuan ZhangZhi-Xin Wang
Apr 4, 2008·The Journal of Biological Chemistry·Matthew J RardinJack E Dixon
Apr 24, 2012·Molecular and Cellular Endocrinology·Bin FengHaiyan Xu
Jan 25, 2002·Annual Review of Pharmacology and Toxicology·Zhong-Yin Zhang
Dec 10, 1999·The Journal of Biological Chemistry·B Zhou, Z Y Zhang
Mar 3, 2007·Biochemistry·Johannes Rudolph
Dec 24, 2003·Biochemistry·Youngjoo KimJohn M Denu

❮ Previous
Next ❯

Related Concepts

Related Feeds

ASBMB Publications

The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.