Abstract
Abstract 2-Keto-4-hydroxyglutarate aldolase has been purified to homogeneity from extracts of Escherichia coli grown on nutrient broth. Values of 62,500 and 64,000, respectively, are obtained for the molecular weight of the enzyme by the procedures of gel filtration and sucrose density gradient centrifugation. The aldolase has a pH optimum of 8.6 and does not appear to require divalent metal ions for activity. p-Mercuriphenylsulfonate, iodoacetate, and N-ethylmaleimide partially inhibit enzymatic activity. The aldolase from this source preferentially cleaves or forms the l isomer of 2-keto-4-hydroxyglutarate. When the enzyme is tested with a number of compounds as substrate, it catalyzes the cleavage of only 2-keto-4-hydroxybutyrate and oxalacetate besides 2-keto-4-hydroxyglutarate. A higher degree of specificity is manifested for binding analogs of pyruvate than of glyoxylate, as determined by the extent of enzyme inactivation when the aldolase is incubated with analog compounds in the presence of NaBH4. Extensive loss of aldolase activity occurs when the enzyme is incubated with either pyruvate or glyoxylate in the presence of NaBH4, and 1 mole of 14C-labeled substrate is stably bound per mole of enzyme. Competition studies indicate that pyruvate, glyoxylate, and 2-keto-4-hydroxyglutarate all bind at the same active site of the aldolase. Incubation of 2-keto-4-hydroxyglutarate aldolase with glyoxylate in the presence of cyanide also causes irreversible loss of enzymatic activity, suggesting the formation of a stable aminonitrile with this substrate. Under these conditions, approximately 1 mole of [14C]glyoxylate is bound per mole of aldolase. Irreversible inactivation of aldolase activity by cyanide is not observed with pyruvate. The pure aldolase from E. coli has a higher relative level of β-decarboxylase activity toward oxalacetate than does the enzyme obtained from extracts of bovine liver. Both aldolase and β-decarboxylase activities are lost when the E. coli aldolase is treated with glyoxylate or pyruvate in the presence of NaBH4, suggesting that the same active site participates in both reactions.
Highlights
When the enzyme is tested with a number of compounds as substrate, it catalyzes the cleavage of only 2-keto-4-hydroxybutyrate and oxalacetate besides 2-keto-4-hydroxyglutarate
Essentially all work concerning the metabolic role and enzymology of KHG-aldolase has been done with rat and bovine liver preparations [4,5,6,7,8,9,10,11,12,13, 16,17,18,19]
One objective of the present work, was to obtain KHG-aldolase in pure form from a bacterium and most desirably from a bacterium which could be used for subsequent genetic studies
Summary
When the enzyme is tested with a number of compounds as substrate, it catalyzes the cleavage of only 2-keto-4-hydroxybutyrate and oxalacetate besides 2-keto-4-hydroxyglutarate. Extensive loss of aldolase activity occurs when the enzyme is incubated with either pyruvate or glyoxylate in the presence of NaBH4, and 1 mole of 14C-labeled substrate is stably bound per mole of enzyme. 4-hydroxyglutarate aldolase with glyoxylate in the presence of cyanide causes irreversible loss of enzymatic activity, suggesting the formation of a stable aminonitrile with this substrate. Under these conditions, approximately 1 mole of [14C]glyoxylate is bound per mole of aldolase. /?-decarboxylase activity toward oxalacetate than does the enzyme obtained from extracts of bovine liver. Both aldolase and /3-decarboxylase activities are lost when the E. coli aldolase is treated with glyoxylate or pyruvate in the presence
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