Die Formamidase von Hansenula henricii: Isolierung, Charakterisierung und Regulation multipler Formen

Abstract

Two active formamidases which hydrolyze N-formylkynurenine and N-formylanthranilic acid into formic acid and kynurenine or anthranilic acid, respectively, have been isolated from the yeast Hansenula henricii CCY 38-10-2. The molecular weight of the formamidase I is about 56,000 D calculated from gel filtration (Sephadex G-100) and 54,00 D by SDS polyacrylamide gel electrophoresis, respectively. Formamidase II has an molecular weight of about 28,000 D, estimated by both methods. After boiling of the formamidase I for 5 min in the presence of SDS (1%) and 2-mercaptoethanol (0.1 %) the enzyme is split into a smaller protein, which in SDS polyacrylamide gel electrophoresis as formamidase II. The p H optimum is different for both enzyme forms: 7.0–8.0 for formamidase I and 5.5 –7.0 for formamidase II. Enzyme activities of the two enzymes are inhibited by Zn ++ , Hg ++ , Cu ++ , NaHSO 4 , p-chloromercuribenzoate, iodacetamide, and NaF. Formamidase II was more strongly inhibited by Zn ++ , but formamidase I more by Cu ++ , whereas the other inhibitors did not show differences. The stability against high temperatures of formamidase I is greater than that of formamidase II. The latter is completely inactivated after 40 min at 45°C, while the smaller enzyme shows still 50% of the initial activity under the same conditions. The K m -values for both substrates are nearly identical 1.9 mM and 2.2 mM for formamidase I and formamidase II, respectively. N-formylkynurenine is hydrolyzed 10 times faster than N-formylanthranilic acid by formamidase I, whereas this factor is 3.6 for formamidase II. If the Hansenula yeasts are grown on minimal medium at p H 4.5 with glucose as carbon source in the presence of anthranilic acid, p-aminobenzoic acid, 3-hydroxyanthranilic acid, kynurenine or 3-hydroxykynurenine, the amount of active formamidase decrease markedly. A repression of enzyme synthesis by these substances is not observed if the p H of the glucose medium is maintained at 6.6, if glucose in the medium is replaced by glycerol, or if ammonia in the medium is replaced by asparagine. Although the ratio of formamidase I to formamidase II varied strongly under the different growth conditions it remains constant during repressed enzyme synthesis.