Catch 222, the effects of symmetry on ligand binding and catalysis in R67 dihydrofolate reductase as determined by mutations at Tyr-69

被引:12
作者
Stinnett, LG [1 ]
Smiley, RD [1 ]
Hicks, SN [1 ]
Howell, EE [1 ]
机构
[1] Univ Tennessee, Dept Biochem & Cellular & Mol Biol, Knoxville, TN 37996 USA
关键词
D O I
10.1074/jbc.M404485200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:47003 / 47009
页数:7
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