Purification, characterization and inhibition of dihydropyrimidinase from rat liver.
Dihydropyrimidinase (DHPase) was purified 564-fold over the initial rat liver extract, using heat, ammonium sulfate fractionation, DEAE-Sepharose CL-6B, carboxymethyl-Sepharose CL-6B, hydroxyapatite and Sephacryl S-300 chromatography. The purified enzyme was shown to be homogeneous by gel electrophoresis both in the presence and absence of SDS. Its molecular mass, determined by gel filtration, was 215 kDa and the subunit mass was 54 kDa. DHPase catalyzed the reversible cyclization of 5,6-dihydrouracil (H2Ura) to N-carbamoyl-beta-alanine or 5,6-dihydrothymine (H2Thy) to N-carbamoyl-beta-aminoisobutyric acid. Authentic 5-bromo-5,6-dihydrouracil (BrH2Ura) and commercially available H2Thy were racemic. However, these 5-substituted 5,6-dihydropyrimidines were hydrolyzed by over 96% and 98%, respectively, by DHPase. These results suggest that dihydropyrimidinase has no stereo specificities for 5-substituents of H2Ura. The addition of H2Ura and H2Thy competitively inhibited the enzyme activity against BrH2Ura. However, the addition of N-carbamoyl-beta-alanine or N-carbamoyl-beta-amino-isobutyric acid showed hyperbolic mixed-type inhibition, when BrH2Ura was used as the substrate. The values of the dissociation constants of BrH2Ura, N-carbamoyl-beta-alanine and N-carbamoyl-beta-aminoisobutyric acid were 17 microM, 0.38 mM and 0.38 mM, respectively. DHPase from the rat liver contains 4 mol Zn2+/mol active enzyme, presumably one atom/subunit. Zn2+ also inhibited the hydrolysis of BrH2Ura by the enzyme. The Ki for Zn2+ as an inhibitor of DHPase was 23 microM, and the maximum rate of inactivation was 0.057 min-1 at 37 degrees C. H2Ura and H2Thy protected the enzyme activity from Zn2+ inactivation.
Kikugawa
M
M
Faculty of Nutrition, Kobe-Gakuin University, Japan.
Kaneko
M
M
Fujimoto-Sakata
S
S
Maeda
M
M
Kawasaki
K
K
Takagi
T
T
Tamaki
N
N
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D008962:Models, Theoretical; D008970:Molecular Weight; D051381:Rats; D013379:Substrate Specificity; D015032:Zinc / Q000032:analysis
D017886:Durapatite; D000581:Amidohydrolases; C020047:dihydropyrimidinase; D015032:Zinc