Purification and properties of 2-pyrone-4,6-dicarboxylate hydrolase.
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-dicarboxylate, 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. Km values are 87 and 26 microM 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 HgCl2 and p-chloromercuribenzoate strongly inhibit the enzyme activity.[1]References
- Purification and properties of 2-pyrone-4,6-dicarboxylate hydrolase. Maruyama, K. J. Biochem. (1983) [Pubmed]
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