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

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 character ization 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 K-cat/K-m parameter (apparent pK(a) values 5.1 and 6.6) and revealed a pH-independent k(cat) 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 (k(cat) = 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.