Trimethoprim binding to Lactobacillus casei dihydrofolate reductase: a 13C NMR study using selectively 13C-enriched trimethoprim.

Abstract

We have measured the 13C chemical shifts for trimethoprim molecules selectively enriched with 13C at the 2-, 4-, 5-, 6-, and 7-positions and the p-OCH3 position in their complexes with Lactobacillus casei dihydrofolate reductase in the presence and absence of coenzyme analogues. The C2 carbon shifts indicate that the pyrimidine ring is protonated at N1 in all the complexes of trimethoprim with the enzyme and coenzymes and in each case the pyrimidine ring is binding in a similar way to that of the corresponding part of methotrexate in the enzyme-methotrexate complex. The C6 carbon of trimethoprim shows a large upfield shift in all complexes (3.51 to 4.70 ppm) but no shift in the complex of 2,4-diaminopyrimidine with the enzyme: these shifts probably arise from steric interactions between the C1' and C2' carbons and the H6 proton, which approach van der Waals contact in the folded conformation adopted by trimethoprim when bound to the enzyme. The large shift observed for C6 in all complexes indicates that the basic folded conformation is present in all of them. A comparison of the 13C shifts in the enzyme-trimethoprim-NADPH complex with those in the enzyme-trimethoprim binary complex shows substantial changes even for carbons such as C6 and p-OCH3 (0.46 and -0.36 ppm, respectively), which are remote from the coenzyme: these are caused by ligand-induced conformational changes that may involve displacement of the helix containing residues 42-49.(ABSTRACT TRUNCATED AT 250 WORDS)