PYRIMIDINE-DEGRADING ENZYMES - PURIFICATION AND PROPERTIES OF BETA-UREIDOPROPIONASE OF EUGLENA-GRACILIS

In photoorganotrophically grown, mid-log phase cells of Euglena gracilis, enzymes of pyrimidine degradation including uracil reductase, dihydrouracil dehydrogenase, dihydropyrimidinase, and β-ureidopropionase, were detected in a crude extract. β-Ureidopropionase (N-carbamoyl-β-alanine amidohydrolase, EC 3.5.1.6) was purified 100-fold by heat treatment, ammonium sulphate fractionation and chromatography using Sepharose 6B and DEAE-Sephadex A-25. The enzyme follows Michaelis-Menten kinetics (Km of β-ureidopropionase for β-ureidopropionate 3.8·10-5 M, Hill coefficient n = 1). Other enzyme properties are: pH optimum 6.25, temperature optimum 60°C, stimulation by Mg2+, inhibition by Cu2+, Mr ≈ 1.5-2·106. β-Ureidoisobutyrate, the intermediate of thymine degradation, and β-ureidopropionate are competing substrates of β-ureidopropionase (Ki = Km of β-ureidopropionase for β-ureidoisobutyrate 1.8·10-5 M). Structural analogues of β-ureidopropionate, isobutyrate and propionate are competitive inhibitors (Ki of β-ureidopropionase 0.3 and 0.16 mM, respectively). There were no indications of regulatory function of β-ureidopropionase in pyrimidine degradation. © 1979.