title: Purification and kinetic characterization of the mitogen-activated protein kinase phosphatase rVH6.
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 supported the assignment of residues 134-381 to the core catalytic domain of rVH6. These results demonstrate that the NH2 terminus of rVH6 (residues 1-133) is not required for full enzyme activity and comprises a separate domain that may play a distinct physiological function.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Wiland
authors.forename: J M
authors.initials: JM
authors.lastname: Denu
authors.forename: R J
authors.initials: RJ
authors.lastname: Mourey
authors.forename: J E
authors.initials: JE
authors.lastname: Dixon
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000302:isolation & purification*; D016415:Sequence Alignment
chemical_list: D017027:Protein Tyrosine Phosphatases