PURIFICATION AND KINETIC CHARACTERIZATION OF THE MITOGEN-ACTIVATED PROTEIN-KINASE PHOSPHATASE RVH6

The dual specificity protein-tyrosine phosphatase rVH6 belongs to a su bfamily of enzymes that have in vivo and in vitro catalytic activity a gainst mitogen-activated protein kinases, A method was developed for t he expression and efficient purification of recombinant rVH6 in quanti ties sufficient for physical and kinetic character ization of the enzy me. Matrix-assisted laser desorption mass spectrometry verified the ma ss of purified rVH6 to be 43,500 +/- 150, and NH2-terminal sequence an alysis confirmed the predicted amino acid sequence. Kinetic characteri zation of full length rVH6 identified the critical ionizations involve d in the K-cat/K-m parameter (apparent pK(a) values 5.1 and 6.6) and r evealed 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 134-3 81 of rVH6 were expressed, purified, and characterized enzymatically. Kinetic analysis revealed that the truncated enzyme exhibited a turnov er value similar to that of the full-length enzyme (k(cat) = 0.017 s(- 1)), with p-nitrophenyl phosphate as substrate, Secondary structure pr ediction and molecular modeling of rVH6 based on the x-ray structure o f the dual specificity protein tyrosine phosphatase, VHR, further supp orted the assignment of residues 134-381 to the core catalytic domain of rVH6, These results demonstrate that the NH2 terminus of rVH6 (resi dues 1-133) is not required for full enzyme activity and comprises a s eparate domain that may play a distinct physiological function.