title: Species-specific irreversible inhibition of Neisseria gonorrhoeae dihydrofolate reductase by a substituted 2,4-diamino-5-benzylpyrimidine.
abstract: Neisseria gonorrhoeae dihydrofolate reductase undergoes a time-dependent, irreversible inactivation by 2,4-diamino-5-[3,5-dimethoxy-4-(p-bromoacetamidophenoxy)benzyl] pyrimidine. The kinetics of inactivation are consistent with the reversible formation of an enzyme-inhibitor complex followed by covalent binding to the enzyme. The reversible component is competitive with dihydrofolate and has an inhibitor binding constant of 10 nM. Irreversible inactivation proceeds as a pseudo first-order process with a minimum inactivation half-time of 20 min and a Ki of 28 nM. Using radiolabeled inhibitor, it was shown that approximately 1 mol of ligand was covalently bound to the enzyme/mol of methotrexate binding site when the enzyme was completely inhibited. Radiolabeled inhibitor remained associated with the enzyme following denaturation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyanogen bromide cleavage of the 14C-labeled enzyme-inhibitor complex yielded only one radioactive polypeptide, and sequence determinations showed that His-25 was modified by covalent attachment of the inhibitor. When dihydrofolate reductases from Lactobacillus casei, Streptococcus faecium, Escherichia coli, SR-1 rodent lymphoma, and chicken liver were tested with the affinity label, only the L. casei enzyme showed a time-dependent increase in inhibition. These data, along with comparisons of known amino acid sequences and x-ray crystal structures, were used to make predictions concerning the three-dimensional conformation of the gonococcal enzyme.
mesh_terms: D005493:Folic Acid Antagonists; D007700:Kinetics; D008433:Mathematics; D009344:Neisseria gonorrhoeae / Q000201:enzymology*; D011743:Pyrimidines / Q000494:pharmacology*; D013045:Species Specificity; D014295:Trimethoprim / Q000031:analogs & derivatives / Q000494:pharmacology; D014961:X-Ray Diffraction
chemical_list: D005493:Folic Acid Antagonists; D011743:Pyrimidines; C031792:2,4-diamino-5-benzylpyrimidine; C043054:2,4-diamino-5-((3,5-dimethoxy-4-(4-bromoacetamidophenoxy)benzyl)pyrimidine); D014295:Trimethoprim