A Structural Role for Glutamine 214 in Human Thymidylate Synthase

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Abstract
Studies of the crystal structures of thymidylate synthase (TS) have revealed that a kink is present in β-sheets that form the core of the enzyme. The β-kink is proposed to serve as a “hinge” during conformational changes that occur in the enzyme after ligand binding at the active site. A residue in one of the β-bulges that form the kink, glutamine at position 214 of human TS, is highly conserved in all TSs and is postulated to interact with nucleotide ligands that bind at the active site. To examine the role of this residue, glutamine at position 214 was replaced by residues that differ in volume, hydrophobicity, electrostatic charge, and hydrogen bonding potential. Genetic complementation studies utilizing a TS-deficient bacterial strain revealed that residues with large side chain volumes or that are prohibited in β-bulges created loss of function proteins. Kinetic studies indicated that residue hydrophobicity is not correlated with catalytic activity. Residues that are predicted to alter the charge at position 214 created enzymes with kcat/Km values at least 103 lower than those of the wild type. Kinetic and ligand binding studies indicated that residue 214 is involved in nucleotide binding; however, hydrogen bonding potential does not contribute significantly to nucleotide binding energy. The data are consistent with the hypothesis that residue 214 is involved in maintaining the enzyme in a conformation that facilitates nucleotide binding and catalysis.