tailieunhanh - Báo cáo khoa học: Tyr235 of human cytosolic phosphoenolpyruvate carboxykinase influences catalysis through an anion–quadrupole interaction with phosphoenolpyruvate carboxylate

Tyr235 of GTP-dependent phosphoenolpyruvate (PEP) carboxykinase is a fully invariant residue. The aromatic ring of this residue establishes an energetically favorable weak anion–quadrupole interaction with PEP carboxylate. The role of Tyr235 in catalysis was investigated via kinetic analysis of site-directed mutagenesis-derived variants. | ễFEBS Journal Tyr235 of human cytosolic phosphoenolpyruvate carboxykinase influences catalysis through an anion-quadrupole interaction with phosphoenolpyruvate carboxylate Lakshmi Dharmarajan1 2 Christopher L. Case1 3 Pete Dunten4 and Biswarup Mukhopadhyay1 2 3 5 1 Virginia Bioinformatics Institute Virginia Polytechnic Institute and State University Blacksburg VA USA 2 Genetics Bioinformatics and ComputationalBiology PhD Program Virginia Polytechnic Institute and State University Blacksburg VA USA 3 Department of Biochemistry Virginia Polytechnic Institute and State University Blacksburg VA USA 4 Stanford Linear Accelerator Center Menlo Park CA USA 5 Department of BiologicalSciences Virginia Polytechnic Institute and State University Blacksburg VA USA Keywords catalysis human PEPCK kinetics site-directed mutagenesis Tyr235 Correspondence B. Mukhopadhyay Virginia Bioinformatics Institute Bioinformatics I Virginia Polytechnic Institute and State University Washington Street 0477 Blacksburg VA 24061 USA Fax 540 231 2606 Tel 1 540 231 8015 E-mail biswarup@ Present address Yale University Schoolof Medicine Section of MicrobialPathogenesis New Haven CT USA Received 1 August 2008 revised 14 September 2008 accepted 24 September 2008 doi Tyr235 of GTP-dependent phosphoenolpyruvate PEP carboxykinase is a fully invariant residue. The aromatic ring of this residue establishes an energetically favorable weak anion-quadrupole interaction with PEP carboxylate. The role of Tyr235 in catalysis was investigated via kinetic analysis of site-directed mutagenesis-derived variants. The Y235F change lowered the apparent Km for PEP by about six-fold raised the apparent Km for Mn2 by about 70-fold and decreased oxaloacetate OAA -form-ing activity by about 10-fold. These effects were due to an enhanced anion-quadrupole interaction between the aromatic side chain at position 235 which now lacked a hydroxyl group and PEP carboxylate which probably .