Crystallographic structure determination of bovine heart and human cytosolic phosphotyrosyl phosphatase
Abstract
Bovine heart and human cytosolic phosphotyrosyl protein phosphatase-isoenzyme A (BHPTP and HCPTPA respectively) belong to the low molecular weight PTPase family. The enzymes in this class have 157 amino acid residues and molecular weights of 18,000. The sequence identity between BHPTP and HCPTPA is 85%. The three dimensional structures of BHPTP and HCPTPA represent the first two structures of this class of enzymes. BHPTP crystallized in the monoclinic space group C2 with unit cell parameters a = 95.3 A, b = 43.3 A, c = 41.2 A, and $\beta$ = 113.4$\sp\circ$. Native data to 2.2 A were collected on a Siemens analytical multiwire area detector with an overall R$\rm\sb{sym}$ of 6.0%. Heavy atom derivative data to 3.0 A were also collected on the Siemens area detector. MIR phases were generated based on four heavy atom derivatives with a FOM of 0.68. The initial model could be built directly from a 3 A electron density map, and has been refined with XPLOR and TNT to 2.2 A with an R-factor of 16.6%. HCPTPA crystallized in the orthorhombic space group P2$\sb1 2\sb1 2\sb1$ with unit cell parameters a = 34.1 A, b = 56.6 A, c = 97.3 A. Native data to 2.2 A were collected on an image plate detector (R-axis II system) with an overall R$\rm\sb{sym}$ of 7.7% The structure of HCPTPA was solved by molecular replacement using the refined BHPTP structure as the model. The final refined HCPTPA structure has an R-factor of 18.1%. The structures of BHPTP and HCPTPA appear quite similar. They contain a central four-strand parallel $\beta$ sheet with flanking $\alpha$-helices on both sides. The overall topology falls into the "Rossmann fold" category, with the active site of the molecule located at the C-terminal end of the sheet. Initial correlation of biochemical data with the structure reveals the location of the two active site cysteines, as well as one histidine and at least one of the arginine residues implicated in the enzymatic reaction. In addition, an aspartic acid residue (Asp 129) has been proposed to be the proton donor based on its position at the active site.
Degree
Ph.D.
Advisors
Stauffacher, Purdue University.
Subject Area
Biochemistry|Biophysics
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.