Effect of a single amino acid substitution on Escherichia coli dihydrofolate reductase catalysis and ligand binding.
The two isozymes of dihydrofolate reductase (Forms 1 and 2) from, a Trimethoprim-resistant strain of Escherichia coli (RT500) were separated and purified to homogeneity using a simple procedure based on differential elution from a Methotrexate affinity column. The complete amino acid sequence of the Form 2 isozyme was determined, and it differs from that of Form 1 in only one position. Residue 28 is arginine in Form 2 and leucine in Form 1. However, the isozymes differ greatly in their binding and kinetic properties. Equilibrium dialysis studies showed the Trimethoprim dissociation constants of Form 2 are about 50-fold greater than those of Form 1 in both the binary complex and the ternary complex with NADPH. Similarly, the Methotrexate dissociation constant of Form 2 is about 10-fold greater than that of Form 1. The two isozymes also differ in their turnover numbers at pH 7 (Form 1 is 10-fold more active) and inhibition by divalent cations. Form 1 is extremely sensitive to BaCl2 (50% inhibition at 0.5 mM), whereas Form 2 is much less sensitive (50% inhibition at 60 mM). In the presence of 10 mM BaCl2, Form 1 has the functional characteristics of Form 2. Its turnover number is decreased, its Trimethoprim Ki is increased, and the shape of its pH-activity profile is identical with that of Form 2. The x-ray structures and amino acid sequences of several bacterial dihydrofolate reductases indicate that Asp-27 is important in inhibitor binding and may be involved in catalysis. The present data provide kinetic evidence for this hypothesis, and it is proposed that almost all the unusual characteristics of Form 2 are the direct result of a charge interaction between Arg-28 and Asp-27. A similar interaction between Ba2+ and the Asp-27 of Form 1 can result in an enzyme complex that is kinetically similar to Form 2.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014295:Trimethoprim / Q000494:pharmacology
D000596:Amino Acids; D007527:Isoenzymes; D008024:Ligands; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase