TAILIEUCHUNG - Báo cáo khoa học: Effect of valine 106 on structure–function relation of cytosolic human thymidine kinase Kinetic properties and oligomerization pattern of nine substitution mutants of V106

Information on the regulation and structure–function rela-tion of enzymes involved in DNA precursor synthesis is pivotal, as defects in several of these enzymes have been found to cause depletion or deletion of mitochondrial DNA resulting in , the effect of aminoacid106 on the enzymatic properties of the cell-cycle-regulated human cytosolic thymidine kinase 1(TK1) is investigated. | Eur. J. Biochem. 271 2248-2256 2004 FEBS 2004 doi Effect of valine 106 on structure-function relation of cytosolic human thymidine kinase Kinetic properties and oligomerization pattern of nine substitution mutants of V106 Hanne Frederiksen t Dvora Berensteint and Birgitte Munch-Petersen Department of Life Sciences and Chemistry Roskilde University Denmark Information on the regulation and structure-function relation of enzymes involved in DNA precursor synthesis is pivotal as defects in several of these enzymes have been found to cause depletion or deletion of mitochondrial DNA resulting in severe diseases. Here the effect of amino acid 106 on the enzymatic properties of the cell-cycle-regulated human cytosolic thymidine kinase 1 TK1 is ir v esliiiated. On the basis of the previously observed profound differences between recombinant TK1 with Val106 V106WT and Met106 V106M in catalytic activity and oligomerization pattern we designed and characterized nine mutants of amino acid 106 differing in size conformation and polarity. According to their oligomerization pattern and thymidine kinetics the TK1mutants can be divided into two groups. Group I V106A V106I and V106T behaves like V106WT in that pre-assay exposure to ATP induces reversible transition from a dimer with low catalytic activity to a tetramer with high catalytic activity. Group II V106G V106H V106K V106L and V106Q behaves like V106M in that they are permanently high activity tetramers irrespective of ATP exposure. We conclude that size and conformation of amino acid 106 are more important than polarity for the catalytic activity and oligomerization of TK1. The role of amino acid 106 and the sequence surrounding it for dimer-tetramer transition was confirmed by cloning the putative interfars fragment of human TK1and investigating its oligomerization pattern. Keywords dimer-tetramer formation enzyme kinetics enzyme mutants structure-function relation thymidine kinase. Enzymes

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