| Abstract
| - Studies of the crystal structures of thymidylatesynthase (TS) have revealed that a kink ispresent in β-sheets that form the core of the enzyme. Theβ-kink is proposed to serve as a “hinge”during conformational changes that occur in the enzyme after ligandbinding at the active site. A residuein one of the β-bulges that form the kink, glutamine at position 214of human TS, is highly conserved inall TSs and is postulated to interact with nucleotide ligands that bindat the active site. To examine therole of this residue, glutamine at position 214 was replaced byresidues that differ in volume, hydrophobicity,electrostatic charge, and hydrogen bonding potential. Geneticcomplementation studies utilizing a TS-deficient bacterial strain revealed that residues with large side chainvolumes or that are prohibited inβ-bulges created loss of function proteins. Kinetic studiesindicated that residue hydrophobicity is notcorrelated with catalytic activity. Residues that are predicted toalter the charge at position 214 createdenzymes with kcat/Kmvalues at least 103 lower than those of the wild type.Kinetic and ligand bindingstudies indicated that residue 214 is involved in nucleotide binding;however, hydrogen bonding potentialdoes not contribute significantly to nucleotide binding energy.The data are consistent with the hypothesisthat residue 214 is involved in maintaining the enzyme in aconformation that facilitates nucleotide bindingand catalysis.
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