Purification and Properties of 2-Pyrone-4, 6-Dicarboxylate Hydrolase

Abstract

A hydrolase which catalyzes specifically the interconversion between 2-pyrone-4, 6-dicarboxylate and 4-oxalmesaconate was purified about 410-fold with a 16% yield from cell-free extracts of Pseudomonas ochraceae grown with phthalate. Upon disc gel electrophoresis, the enzyme preparation gave a single band which was coincident with the enzyme activity. The molecular weight of the enzyme was estimated to be 31, 000 by gel filtration on Sephadex G-75 and 33, 000 by sodium dodecyl sulfate gel electrophoresis. The isoelectric point of the enzyme was determined to be at pH 5.49 by isoelectric focusing. The enzyme is specific for 2-pyrone-4, 6-dicarbox-ylate, and various other lactones did not serve as substrates. The stoichiometry of 2-pyrone-4, 6-dicarboxylate hydrolysis, 4-oxalmesaconate formation and proton production was approximately 1:1:1. The optimum pHs are 8.5 and 6.0 for hydrolysis and synthesis of 2-pyrone-4, 6-dicarboxylate, respectively. K_m values are 87 and 26 μM for 2-pyrone-4, 6-dicarboxylate and 4-oxalmesaconate, respectively. At pH 8.5, the ratio of 4-oxalmesaconate to 2-pyrone-4, 6-dicarboxylate at equilibrium is about 2.2. Thiol reagents such as HgCI_ and p-chloromercuribenzoate strongly inhibit the enzyme activity.