title: Thymidylate synthetase from Diplococcus pneumoniae, properties and inhibition by folate analogs.
abstract: Thymidilate synthetase (methylenetetrahydrofolate:dUMP C-methyltransferase) in crude extract from Diplococcus pneumoniae exhibits a partial but variable requirement for Mg-2+ depending upon the buffer. Optimum Mg-2+ concentration is between 0.014 and 0.02 M. The optimum pH for activity in a variety of buffers occurred as a broad peak between 7.0 and 7.7. In Tris/acetate buffer, but not in potassium phosphate buffer, the pH optimum was different in the presence and absence of Mg-2+. Methylation of uridylate, cytidylate and deoxycytidylate could not be demonstrated over a pH range of 5.0-8.0. The enzyme exhibited an apparent Km for deoxyuridylate of 3.08 - 10-5 M and an apparent Km for L-(+)(minus)-5,10-methylene tetrahydrofolate of 2.66 - 10-4 M. During molecular-sieve chromatography and sucrose density-gradient centrifugation, the enzyme was detectable only as a single catalytically active form of Mr 34 000-38 000. 2,4-Diamino quinazoline antifolates were better competitive inhibitors (Ki = 3-8 -10-6 M) of thymidylate synthetase than 2,4-diamino pteridines (Ki = 3- 10-5 M). 2-Amino-4-hydroxy-quinazolines were the best inhibitors (Ki = 1.3-2.9 - 10-6 M). All of the 2,4-diamino quinazolines and pteridines inhibited dihydrofolate reductase from D. pneumoniae in a nearly stoichiometric fashion (Ki = less than 10-10 M). The 2-amino-4-hydroxy-quinazolines were poor inhibitors of this enzyme (Ki = 10=5 M).
mesh_terms: D002850:Chromatography, Gel; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008780:Methyltransferases / Q000378:metabolism*; D008970:Molecular Weight; D011799:Quinazolines / Q000494:pharmacology; D013296:Streptococcus pneumoniae / Q000187:drug effects / Q000201:enzymology*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013940:Thymidylate Synthase / Q000378:metabolism*
chemical_list: D005493:Folic Acid Antagonists; D011799:Quinazolines; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D008780:Methyltransferases; D013940:Thymidylate Synthase; D008274:Magnesium