"Catch 222," the effects of symmetry on ligand binding and catalysis in R67 dihydrofolate reductase as determined by mutations at Tyr-69.
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 Km values for both ligands and a 2-fold rise in the kcat 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 Kd and Km 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 Km 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.
Stinnett
Lori G
LG
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, USA.
Smiley
R Derike
RD
Hicks
Stephanie N
SN
Howell
Elizabeth E
EE
D001665:Binding Sites; D002151:Calorimetry; D002384:Catalysis; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry*
D007477:Ions; D008024:Ligands; D014443:Tyrosine; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase; D007930:Leucine