Characteristics of NADPH-dependent thymidylate synthetase purified from Streptomyces aureofaciens.
NADPH-dependent thymidylate synthetase from Streptomyces aureofaciens has been purified to homogenity by a two-step chromatographic procedure including anion-exchange chromatography and affinity chromatography on methotrexate-Sepharose 4B. The enzyme was purified 1025-fold with a 34% yield. Basic characteristics of the enzyme were determined: molecular weight of the enzyme subunit (28,000), pH and temperature optimum, effect of cations, dependency on reducing agents, Km values for dUMP, mTHF, and NADPH (3.78, 21.1, and 38.9 microM, respectively), and inhibition effect of 5-FdUMP. Binding studies revealed the enzyme mechanism to be ordered sequential: dUMP bound before mTHF. S. aureofaciens thymidylate synthetase exhibits an absolute requirement for NADPH for the enzyme activity--a unique feature not displayed by any of the thymidylate synthetases isolated so far. NADPH is not consumed during enzyme reaction, indicating its regulatory role. The properties of S. aureofaciens thymidylate synthetase show that it is a monofunctional bacterial enzyme.
Gálová
M
M
Slovak Academy of Sciences, Institute of Molecular Biology, Bratislava.
Koptidesová
D
D
Rusznáková
D
D
Racay
P
P
Kollárová
M
M
D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005468:Fluorodeoxyuridylate / Q000494:pharmacology; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D013304:Streptomyces aureofaciens / Q000201:enzymology*; D013762:Tetrahydrofolate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000302:isolation & purification* / Q000378:metabolism*
D005468:Fluorodeoxyuridylate; D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase