A STRUCTURAL ROLE FOR GLUTAMINE-214 IN HUMAN THYMIDYLATE SYNTHASE

Studies of the crystal structures of thymidylate synthase (TS) have re vealed that a kink is present in beta-sheets that form the core of the enzyme. The beta-kink is proposed to serve as a ''hinge'' during conf ormational changes that occur in the enzyme after ligand binding at th e active site. A residue in one of the beta-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 th e active site. To examine the role of this residue, glutamine at posit ion 214 was replaced by residues that differ in volume, hydrophobicity , electrostatic charge, and hydrogen bonding potential. Genetic comple mentation studies utilizing a TS-deficient bacterial strain revealed t hat residues with large side chain volumes or that are prohibited in b eta-bulges created loss of function proteins. Kinetic studies indicate d that residue hydrophobicity is not correlated with catalytic activit y. Residues that are predicted to alter the charge at position 214 cre ated enzymes with k(cat)/K-m values at least 10(3) lower than those of the wild type. Kinetic and ligand binding studies indicated that resi due 214 is involved in nucleotide binding; however, hydrogen bonding p otential does not contribute significantly to nucleotide binding energ y. The data are consistent with the hypothesis that residue 214 is inv olved in maintaining the enzyme in a conformation that facilitates nuc leotide binding and catalysis.