User:Bekidl

R67 DHFR is a type II R-plasmid-encoded DHFR without genetically and structurally relation to the chromosomal DHFR. It is a homotetramer that possesses the 222 symmetry with a single active site pore that is exposed to solvent[null .][QT1]  This symmetry of active site results in the different binding mode of the enzyme: It can bind with two dihydrofolate (DHF) molecules or two NADPH molecules, or one substrate plus one, but only the latter one has the catalytical activity[null .][QT2]  Compare with E. coli chromosomal DHFR, it has higher Km in binding dihydrofolate (DHF) and NADPH. The much lower catalytical kinetics show that hydride transfer is the rate determine step rather than product (THF) release[null .][QT3]

Given the configuration of its active-site, it is unlikely that R67 DHFR has been able to acquire a residue similar to D27 in Ec chromosomal DHFR. This limitation occurs because addition of a single mutation (in the gene) that might activate DHF will result in four mutations per active site. While addition of a general acid might activate DHF in one site, it would more than likely impede NADPH binding in the other symmetry-related site(s). To identify any potential groups involved in catalysis the pH profile of a H62C R67 DHFR was monitored[null .] [QT4] This mutant stabilized the active tetramer from pH 4–9 by disulfidebond formation. Its pH profile resembled that of the Ec D27S DHFR mutant, with increasing activity as the pH was lowered. This behavior is consistent with the use of protonated DHF as the productive substrate and no contributions from acidic groups in the enzyme. Raman difference measurements also show no indication of bound protonated DHF at pH 5.3; this suggests that the active site environment of R67 DHFR does not greatly alter the pKa of N5 in DHF from the solution value of 2.59[null .][QT5]  This observation is consistent with the active site’s being large and accessible to solvent. Interligand interactions appear quite important to the R67 DHFR reaction. Various pairs of ligands compete for binding to the active-site pore with the DHF·DHF and NADPH·NADPH complexes being nonproductive. From both crystallography and NMR studies, the 2DHF/folate molecules stack near the center of the pore in a manner perhaps similar to their solution dimeric structure[null .][QT6]  From docking studies as well as monitoring interligand NOEs, the folate·NADP+ complex also appears to involve stacking between the nicotinamide and pteridine rings. Thus, ring stacking might be strongly correlated with the positive cooperativity associated with 2DHF/folate and NADPH·folate complex formation. Does R67 DHFR also play a role in this cooperativity? From studies of asymmetric Q67H mutants, Q67 at the center of the pore helps discriminate between the various complexes, with a strong preference for the productive NADPH·DHF pair[null .][QT7]  This result suggests that an interplay between the wt protein and the interligand complexes provides a funnel towards transition-state formation.

When ternary-complex formation is studied by ITC, the DH value associated with addition of folate to wt and mutant R67 DHFR·NADPH complexes shows a potential linear correlation with catalytic efficiency. [QT1]Narayana, N., Matthews, D. A., Howell, E. E., and Nguyen-huu, X. (1995) A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site, Nat. Struct. Biol. 2, 1018-1025.

[QT2]Bradrick, T. D., Beechem, J. M., and Howell, E. E. (1996) Biochemistry 35, 11414-11424.

[QT3]H. Park, P. Zhuang, R. Nichols, E. E. Howell, J. Biol. Chem. 1997, 272, 2252 –2258.

[QT4]H. Park, P. Zhuang, R. Nichols, E. E. Howell, J. Biol. Chem. 1997, 272, 2252 –2258.

[QT5]H. Deng, R. Callender, E. E. Howell, J. Biol. Chem. 2001, 276, 48956 – 48960.

[QT6]D. Li, L. A. Levy, S. A. Gabel, M. S. Lebetkin, E. F. DeRose, M. J. Wall, E. E. Howell, R. E. London, Biochemistry 2001, 40, 4242– 4252.

[QT7]R. D. Smiley, L. G. Stinnett, A. M. Saxton, E. E. Howell, Biochemistry 2002, 41, 15664 –15675.