 |
|
Title
|
|
The purification and characterization of a human dual-specific protein tyrosine phosphatase.
|
|
|
Authors
|
|
J.M.Denu,
G.Zhou,
L.Wu,
R.Zhao,
J.Yuvaniyama,
M.A.Saper,
J.E.Dixon.
|
|
|
Ref.
|
|
J Biol Chem, 1995,
270,
3796-3803.
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
An expression and purification method was developed to obtain the recombinant
human dual-specific protein tyrosine phosphatase (PTPase) VHR in quantities
suitable for both kinetic studies and crystallization. Physical characterization
of the homogeneous recombinant protein verified the mass to be 20,500 +/- 100 by
matrix-assisted laser desorption mass spectrometry, confirmed the anticipated
NH2-terminal amino acid sequence and demonstrated that the protein exists as a
monomer. Conditions were developed to obtain crystals which were suitable for
x-ray structure determination. Using synthetic diphosphorylated peptides
corresponding to MAP177-189 (mitogen-activated protein) kinase
(DHTG-FLpTEpYVATR), an assay was devised which permitted the determination of
the rate constants for dephosphorylation of the diphosphorylated peptide on
threonine and tyrosine residues. The diphosphorylated peptides are preferred
over the singly phosphorylated on tyrosine by 3-8-fold. The apparent
second-order rate constant kcat/Km for dephosphorylation of phosphotyrosine on
DHTGFLpTEpYVATR was 32,000 M-1 S-1 while dephosphorylation of phosphothreonine
was 14 M-1 S-1 (pH 6). The reaction of DHTGFLpTEpYVATR with VHR is ordered, with
rapid dephosphorylation on tyrosine occurring first followed by slow
dephosphorylation on threonine. Similar results were obtained with
F(NLe)(N-Le)pTPpYVVTR, a peptide corresponding to a MAP kinase-like protein
(JNK1(180-189)) which is involved in the stress response signaling pathway.
|
|
|
|