Masters Theses

Date of Award

12-1991

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Elizabeth E. Howell

Committee Members

John W. Koontz, Engin Serpersu

Abstract

Aspartic acid 27 is conserved in all bacterial dihydrofolate reductases (DHFR) and it serves as a proton donor in the catalytic mechanism (Howell et al., Sci 23:1123). Not surprisingly, the D27S mutant protein has a greatly decreased kcat at pH 7.0. Second site suppressor mutations have been generated in the D27S DHFR gene by genetic selections and site-directed mutagenesis techniques. These mutations partially suppress the effect caused by removal of the proton donor. To investigate whether these individual suppressing mutations have additive effects, a D27S+E17G+D127G+F137S quadruple mutant gene was constructed. The kcat and Km (DHF) of the quadruple mutant DHFR are similar to those of the D27S+F137S double mutant, thus an additive effect is not found in the quadruple mutant DHFR. Additionally, the D27S+T113E+F153S and D27S+T113E+F137S and D27S+T113E+I155N triple mutant DHFR genes were generated to test the relationship between the active site and a β-bulge region. The T113E mutation puts a potential proton donating group in the active site, although at a different position. The triple mutants have different mobilities on a nondenaturing polyacrylamide gel when compared to wild-type and D27S DHFRs, indicating altered conformations. The kcat and Km values of the D27S+T113E+F153S triple mutant DHFR are slightly better than those of the D27S+T113E double mutant but they are worse than those of the D27S+F153S double mutant DHFR. The triple mutant DHFR does not show additive effects.

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