Purification et propriétés de la d-mannonate hydrolyase d' Escherichia coli

Abstract

1. 1. d-Mannonate hydrolyase (EC 4.2.1.8), an enzyme of the hexuronate pathway, has been purified by ammonium sulfate precipitation, chromatography on DEAE-cellulose and Sephadex G-200 gel filtration. The overall procedure results in a 61-fold purification with a yield of 34%. 2. 2.Optimal conditions for enzyme assay have been established : glycylglycine buffer at pH 8.3, in the presence of ferrous sulfate and 2-mercaptoethanol as activators and protector. 3. 3.Mannonate hydrolyase is inducible by glucuronate and fructuronate, but not by galacturonate and tagaturonate. It has a maximum activity between pH 7 and 8.5. Among numerous compounds tested, only mannonate is a substrate with a K m of 20 mM. Several inhibitors of the enzyme have been studied, and the following three are competitive inhibitors: d-gluconate, d-mannitol and d-sorbitol. Some intermediates of the degradative hexuronate pathway activate the mannonate hydrolyase: d-glucuronate, d-galacturonate, d-tagaturonate and d-altronate. 4. 4.Thermal inactivation of mannonate hydrolyase in the presence of urea indicates that at last 85% of the activity of the enzyme is homogenous. The protection against heat denaturation by the competitive inhibitor d-gluconate confirms the nature of the inhibition. 5. 5.The enzyme is inactivated by 1 mM p-chloromercuribenzoate. 6. 6.The product of enzyme action on d-mannonate has been identified as 2-keto-3-deoxy- d-gluconate by paper chromatography.