Creation and characterization of asymmetric mutations in R67 dihydrofolate reductases

Author

Heonyong Park

Date of Award

5-1997

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Elizabeth E. Howell

Committee Members

Jorge Churchich, Cynthia Peterson, Solon Georghiou

Abstract

Dihydrofolate reductase (DHFR) catalyzes the NADPH dependent reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8- tetrahydrofolate (THF). DHFR is an essential enzyme for cell growth, since THF is necessary for DNA synthesis and other cellular processes. Trimethoprim (TMP), a DHFR inhibitor, is utilized in the clinical treatment of bacterial infections and malaria.

R67 DHFR, a type II plasmid-encoded DHFR, confers TMP resistance and is structurally and genetically unrelated to chromosomal DHFR. The homo-tetrameric form of R67 DHFR is the active species and it dissociates into relatively inactive dimers at pH≤7. Construction of an H62C mutant R67 DHFR eliminates this pH-dependent dissociation by formation of disulfide bonds at the dimer- dimer interfaces. A pH profile of activity in oxidized H62C R67 DHFR displays increasing activity at lower pH, supporting the previous observation that a proton donor does not occur in the active site pore (Narayana et al., 1995 & Park et al., in press). Accordingly, the model for catalysis of R67 DHFR describes binding and reduction of protonated substrate which contrasts with mechanisms proposed for chromosomally encoded DHFRs, where a proton donor is used to facilitate catalysis.

To evaluate the role of Q67 in ligand binding and catalysis, a Q67H mutant R67 DHFR was constructed. The Q67H mutation has tightened binding to both substrate and cofactor. While NADPH binding displays negative cooperativity, DHF/folate binding displays no cooperativity. From binary and ternary complex experiments, it is observed that a maximum of 2 ligands can bind per active site pore. Due to the 222 symmetry of R67 DHFR, these observations support the hypothesis that NADPH and DHF both bind to the same half pore (presumably with different orientations). Since the Ką values for both NADPH and DHF binding are decreased substantially in the Q67H mutant and the positive cooperativity in DHF binding observed with the wild-type enzyme is lost, both substrate and cofactor inhibition are readily observed.

Asymmetric mutants have been constructed using both gene duplication and H62C disulfide bond engineering techniques. At pH 7, the kinetic behavior of 2Q67H R67 DHFR (possessing 2 Q67H mutations per active site pore) displays substrate and cofactor inhibition. However, the kinetic behavior of 2Q67H R67 DHFR displays no substrate and cofactor inhibition at pH 5. This change in inhibition behavior may be caused by the ability to partition into an alternate kinetic pathway. A pH profile for k_cat in 1Q67H R67 DHFR (possessing 1 Q67H mutation per active site pore) shows no titration from pH 5 to 7, indicating that Q67H is not acting as a proton donor. The kinetic efficiency (k_cat/Km(_NADPH)) of 1Q67H R67 DHFR at pH 7 is 10 fold higher than that of wt R67 DHFR.

Recommended Citation

Park, Heonyong, "Creation and characterization of asymmetric mutations in R67 dihydrofolate reductases. " PhD diss., University of Tennessee, 1997.
https://trace.tennessee.edu/utk_graddiss/9580