Structure and activity studies on mutants of the enzyme dihydrofolate reductase.
thesisposted on 19.11.2015, 09:07 by Janette Ann. Thomas
During the course of this thesis four mutants of Lactobacillus casei dihydrofolate reductase have been studied to determine the role of the individual amino acids in the structure and function of the enzyme. The mutants tryptophan-21→ histidine and threonine-63 → alanine were successfully made by site specific mutagenesis. The mutant aspartate-26 →glutamine was successfully expressed and purified but did not bind as tightly to the methotrexate affinity column as the wild type. This mutant had only 1% of the activity of the wild type enzyme. The threonine-63 →glutamine mutant was successfully expressed and purified. The equilibrium binding constants of substrates and inhibitors binding to binary and ternary enzyme complexes were the same as wild type except the binding of the oxidised coenzyme to the mutant apoenzyme, which was reduced three fold. kcat, and Km for NADPH were unaffected by the mutation, however the apparent pKa of catalysis and the pKa of the isotope effect were both reduced by 0.5 pH units. Nmr analysis revealed no structural changes at residues near the mutation or to the bound coenzyme, however there were small structural affects seen at the substrate binding site, at least 15A away from the mutation. A movement along the backbone of helix-C, from glutamine-43 at the mutation site, to the substrate binding site is hypothesized to cause these affects. Expression of threonine-63 → alanine was only detected when the temperature was at 30°C rather than the usual 40°C. kcat and the binding of substrate and inhibitors were the same as wild type. The binding of coenzyme was reduced by six hundred fold, the rate of association reduced by fifty fold and the rate of dissociation increased by twelve fold. Nmr analysis revealed the adenine and the 2' phosphate moieties of NADPH are affected by the mutation, the adenine ring being further away from residues in the protein. These affects were not simply due to the loss of the hydrogen bond between threonine and the protein but must also be caused by some conformational change in the protein.