A structure—function study of dihydrofolate reductase by protein engineering

Abstract

Several mutants of the enzyme dihydrofolate reductase (DHFR) have been engineered by oligonucleotide-directed mutagenesis of the cloned E. coli gene. The mutations were designed to address specific questions about DHFR structure-function relations that arose from the analysis of the high-resolution structure. Mutations at the active site have revealed that the invariant residue aspartate-27 is involved in substrate protonation, and not in transition-state stabilization as previously thought. The 2.0 Å (1 Å = 10 ^-1 nm = 10 ^-10 m) refined structures of the Asn-27 and Ser-27 mutant enzymes reveal that the enhanced binding observed for the 2,4-diamino pteridine and pyrimidine inhibitors is probably not attributable to the charge interaction between Asp-27 and a protonated N-1 of the inhibitor. Substitution of a cysteine for a proline at position 39 places two sulphydryls within bonding distance, and under certain oxidation conditions they will quantitatively form a disulphide bond. The refined 2.0 Å structures of both reduced and oxidized forms of this mutant show that only minor conformational changes occur for disulphide bond formation. The crosslinked enzyme is significantly more conformationally stable to denaturants such as guanidine hydrochloride and urea.