“Catch 222,” the Effects of Symmetry on Ligand Binding and Catalysis in R67 Dihydrofolate Reductase as Determined by Mutations at Tyr-69

• Published in: The Journal of biological chemistry 2004-11, Vol.279 (45), p.47003-47009
• Main Authors: Stinnett, Lori G, Smiley, R Derike, Hicks, Stephanie N, Howell, Elizabeth E
• Format: Article
• Language: English
• Subjects: Binding Sites; Calorimetry; Catalysis; Dimerization; Escherichia coli - enzymology; Hydrogen-Ion Concentration; Ions; Kinetics; Leucine - chemistry; Ligands; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Mutation; NADP - chemistry; Protein Binding; Protein Structure, Tertiary; Tetrahydrofolate Dehydrogenase - chemistry; Thermodynamics; Tyrosine - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M404485200

R67 dihydrofolate reductase (R67 DHFR) catalyzes the transfer of a hydride ion from NADPH to dihydrofolate, generating tetrahydrofolate. The homotetrameric enzyme provides a unique environment for catalysis as both ligands bind within a single active site pore possessing 222 symmetry. Mutation of one active site residue results in concurrent mutation of three additional symmetry-related residues, causing large effects on binding of both ligands as well as catalysis. For example, mutation of symmetry-related tyrosine 69 residues to phenylalanine (Y69F), results in large increases in K m values for both ligands and a 2-fold rise in the k cat value for the reaction (Strader, M. B., Smiley, R. D., Stinnett, L. G., VerBerkmoes, N. C., and Howell, E. E. (2001) Biochemistry 40, 11344–11352). To understand the interactions between specific Tyr-69 residues and each ligand, asymmetric Y69F mutants were generated that contain one to four Y69F mutations. A general trend observed from isothermal titration calorimetry and steady-state kinetic studies of these asymmetric mutants is that increasing the number of Y69F mutations results in an increase in the K d and K m values. In addition, a comparison of steady-state kinetic values suggests that two Tyr-69 residues in one half of the active site pore are necessary for NADPH to exhibit a wild-type K m value. A tyrosine 69 to leucine mutant was also generated to approach the type(s) of interaction(s) occurring between Tyr-69 residues and the ligands. These studies suggest that the hydroxyl group of Tyr-69 is important for interactions with NADPH, whereas both the hydroxyl group and hydrophobic ring atoms of the Tyr-69 residues are necessary for proper interactions with dihydrofolate.