Site-directed mutagenesis of mouse thymidylate synthase: alteration of Arg44 to Val44 in a conserved loop guarding the active site has striking effects on catalysis and nucleotide binding.
The arginine located at position 44 of mouse thymidylate synthase is in a highly conserved loop that is in close proximity to the active site cleft of the enzyme. Structural analyses have suggested that this arginine forms hydrogen bonds with the alpha-carboxylate of the C-terminal amino acid and the phosphate of the substrate analog, FdUMP (D. A. Matthews, K. Appelt and S. J. Oatley, (1989) Adv. Enz. Reg., 29, 47-60). We have used protein engineering techniques to change this amino acid residue to valine. This alteration leads to large reductions in the ability of the enzyme to form covalent complexes with substrate (dUMP) or inhibitor (FdUMP) and at least a 100-fold reduction in catalytic activity. These observations show that this arginine plays an important role in maintaining catalytic activity of the enzyme.
Zhang
H C
HC
Department of Biochemistry, Ohio State University, Columbus 43210.
Cisneros
R J
RJ
Deng
W L
WL
Johnson
L F
LF
Dunlap
R B
RB
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D005468:Fluorodeoxyuridylate / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D011487:Protein Conformation; D013940:Thymidylate Synthase / Q000235:genetics* / Q000378:metabolism; D014633:Valine
D003856:Deoxyuracil Nucleotides; D015345:Oligonucleotide Probes; D005468:Fluorodeoxyuridylate; D001120:Arginine; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase; D014633:Valine