ИСТИНА

Dihydrofolate reductase (DHFR) catalyses the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate using NADPH as coenzyme. The enzyme is of considerable pharmacological interest being the target for a number of clinically useful antibacterial and antineoplastic drugs. We have obtained high-resolution solution structures for several complexes of Lactobacillus casei DHFR (162 a.a., M.W. ~ 18300 Dalton) formed with the antineoplastic drugs methotrexate, trimetrexate and trimethoprim. The locations of the ligands in the binding sites of enzyme have been examined and several specific interactions between the ligands and surrounding protein residues have been studied in details using quantum mechanical calculations. Protein-backbone dynamics in the binary and ternary complexes of DHFR with trimethoprim and NADPH have been studied using 15N relaxation measurements. Analysis of the NMR relaxation data measured at two different magnetic fields allowed us to obtain information about fast (nanosecond time scale) and slower (millisecond time scale) motions of the protein backbone. Motions of trimethoprim in the active site of the enzyme have also been studied by using dynamic NMR spectroscopy methods. The information obtained from these studies increases our knowledge of the factors which control the specificity of inhibitor-enzyme binding and can be useful both for development of new antifolate drugs and for understanding of the details of the mechanism of catalytic activity of dihydrofolate reductase.