Limited proteolysis of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania tropica.
The structure and activity of the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from the protozoan parasite Leishmania tropica were examined by limited proteolysis with five different endopeptidases. Each reaction resulted in a rapid, time-dependent loss of TS activity and no effect on DHFR activity. The proteolytic products were examined by NaDodSO4/PAGE; each digestion produced a fragment of apparent Mr approximately 35,000, and three of the five digestions generated a fragment of Mr approximately 20,000. Attempts to separate the fragments under nondenaturing conditions failed, suggesting that the proteolyzed protein remains a dimer with the gross structure of the subunits more or less undisturbed. In contrast, kinetic data indicate that some aspects of higher-order structure in the native protein are affected by proteolysis. The fragments (Mr 36,600 and 20,000) generated by Staphylococcus aureus V8 protease were subjected to sequence analysis. Whereas neither the native protein nor the Mr 36,600 fragment yielded an NH2-terminal amino acid, we obtained the sequence of the first 28 amino acids of the Mr 20,000 fragment. This sequence bore strong homology with sequences situated within TS of human, Lactobacillus casei, Escherichia coli, and bacteriophage T4. These and other data indicate that the TS-DHFR polypeptide consists of a DHFR sequence at the blocked NH2-terminal and a TS sequence at the COOH-terminal end of the protein. The region that is the target of the five proteases corresponds to a highly variable region within the sequences of the other four TSs. We suggest that an insertion occurs within the TS-DHFR sequence, positioned on the surface of the protein and quite vulnerable to the action of endopeptidases.
D000595:Amino Acid Sequence; D010450:Endopeptidases; D020689:Exopeptidases; D007895:Leishmania tropica / Q000201:enzymology*; D010447:Peptide Hydrolases; D010455:Peptides / Q000032:analysis; D012689:Sequence Homology, Nucleic Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000032:analysis*; D013940:Thymidylate Synthase / Q000032:analysis*
D010455:Peptides; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D010450:Endopeptidases; D020689:Exopeptidases; D010447:Peptide Hydrolases