Kinetic and structural studies of the low molecular weight protein tyrosine phosphatase from Tritrichomonas foetus
Abstract
Low molecular weight protein tyrosine phosphatases are a family of phosphomonoesterase enzymes found in both prokaryotic and eukaryotic organisms. They are characterized by the presence of the highly conserved CX5R motif and a molecular weight of approximately 18 kDa. Several eukaryotic low molecular weight protein tyrosine phosphatases have been studied to date, and they contain a conserved, high-pKa histidine residue. This residue and another highly conserved serine are hydrogen bonded to a conserved active site asparagine residue located in the phosphate binding loop. These interactions are important for substrate binding and catalysis. In the phosphatase from the bovine parasite Tritrichomonas foetus, the conserved serine is present, but the otherwise highly conserved histidine has been replaced with a glutamine residue. Site-directed mutagenesis, kinetic, and spectroscopic experiments indicated that S37 and Q67 are located near the active site and are important for optimal catalytic function. Importantly, the pH-rate profile of wild-type TPTP revealed a much more clearly defined acidic limb than that which can be observed for other wild-type low molecular weight phosphatases. Nuclear magnetic resonance spectroscopy was used to determine the three dimensional structure of the TPTP enzyme in order to further examine the roles of S37 and Q67 in the active site. The TPTP structure contains a four-strand, parallel β-sheet surrounded by five flanking α-helices. The backbone conformation of the phosphate binding loop is nearly superimposable with that of other tyrosine phosphatases. As expected, both S37 and Q67 are located at the active site. However, they are not within strict hydrogen bonding distance of N14. Based on the kinetic and structural data, the interactions of S37 and Q67 with N14 may be equally important to stabilizing this residue. These findings afford new insight into the factors affecting the pH dependence and catalysis of this family of enzymes.
Degree
Ph.D.
Advisors
Etten, Purdue University.
Subject Area
Biochemistry|Biophysics
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