Masters Theses

Date of Award

12-1992

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Elizabeth E. Howell

Committee Members

Engin Serpersu, Solan Georghiou

Abstract

R67 dihydrofolate reductase (DHFR) is a plasmid encoded DHFR which confers high levels of resistance upon the host cell to the antibacterial agent trimethoprim (TMP). This enzyme shares no sequence or structural homology with the E. coli chromosomal DHFR yet the two enzymes carry out identical reactions. Kinetic pH profiles have shown that the catalytic activity of R67 DHFR is dependent on an acidic titration. From the tetramer crystal structure (Narayana, Matthews, Xuong, personal communication) hydrophobic interactions between symmetry related histidines 62,162,262,362 and tryptophans 38,138,238,338 at the dimer:dimer interfaces appear to be the predominate forces that stabilize the tetramer. Intonation of histidine could destabilize the tetrameric R67 DHFR. To determine if the observed kinetic pH titration is due to the ionization of histidines 62,162,262,362, the stability of tetrameric R67 DHFR has been monitored as a function of pH by gel filtration and fluorescence techniques. Gel filtration studies show that in the pH range 5-8 R67 DHFR reversibly dissociates into dimers. Since Tryptophans 38,138,238,338 also occur at the dimer interface, fluorescence was used to monitor the tetramer-dimer dissociation. A pH linked tetramer-dimer dissociation model is presented and used to analyze the data. In this model, histidines in dimeric R67 DHFR are protonated at low pH, pKa = 6.84. Protonation stabilizes the dimeric form of R67 DHFR and shifts the tetramer-dimer equilibrium, Kd = 9.72 nM, towards dimer. Modification of histidines 62,162,262,362 by diethylpyrocarbonate (DEPC) stabilizes dimeric R67 DHFR and causes a 200-600 fold decrease in catalytic efficiency. Finally, site-directed mutagenesis has been used to replace histidine 62 with cysteine. The disulfide bonded form of the H62C mutant is catalytically active and remains tetrameric at low pH. The results indicate that protonation of histidine is linked to the tetramer-dimer equilibrium and that formation of dimer results in decreased catalytic activity. This decrease in activity is presumably due to loss of the putative active site pore in tetrameric R67 DHFR.

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