[Formamidase--microheterogeneity, catalytic properties and inhibitors (author's transl)].

Formamidase from rat liver proved to be microheterogenous. After preparative isoelectric focusing in density gradient columns, two peaks of formamidase with identical substrate specificity were identified. By analytical focusing in thin layers of polyacrylamide or Sephadex G-75 SF, even five bands could be separated. Their isoelectric points were 4.75, 4.78, 4.82, 4.92 (main band) and 5.11, but their Michaelis constants did not differ significantly (54 to 62 mumol/l). An identical molecular weight of 34700 +/- 3200 for all bands was determined by disc electrophoresis. This value was confirmed by sedimentation analyses (so20,w = 3.00 S) and electrophoresis in the presence of sodium dodecyl-sulfate (Mr 34900 +/- 2300), which only gave a single band. The homogeneity was also confirmed by electrophoresis in the presence of 6M urea. Repeated disc electrophoresis of focusing under native conditions with single, isolated formamidases again resulted in different bands which were identified, not only by Coomassie Blue, but also by their hydrolytic cleavage of naphthyl acetate. Formamidase showed neither proteolytic nor asparagine-amidohydrolase activity and oligosaccharide conjugates were not detectable. Ampholytes, buffer ions, pH and peroxodisulfate did not affect the heterogeneity. "Initial burst" measurements with diethyl(4-nitrophenyl) phosphate yielded an equivalent weight of 36,300. Formylkynurenine reduced this inhibition very effectively. Thus, an extraordinary reactive serine residue appeared to be located in the catalytic site of formamidase. A participation of sulfhydrylgroups in the inactivating reaction of arsenite was excluded although two such groups were detected by 5,5'-dithiobis(2-nitrobenzoic acid). N-Bromosuccinimide reacted primarily with one of the nine tryptophan residues without loss of enzymatic activity, but a 18.6-fold excess of this reagent resulted in a complete loss of activity. The reaction rates of the most effective inhibitors and of the protective action of formylkynurenine were determined. Thus, formamidase must clearly be distinguished from typical serine esterases and proteases.