2-Keto-4-hydroxyglutarate aldolase: purification and characterization of the homogeneous enzyme from bovine kidney.
2-Keto-4-hydroxyglutarate aldolase, which catalyzes the reversible cleavage of 2-keto-4-hydroxyglutarate, yielding pyruvate plus glyoxylate, has been purified from extracts of bovine kidney to apparent homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration chromatography, sucrose density gradient centrifugation, and meniscus depletion sedimentation equilibrium experiments. The enzyme from this source has a native and a subunit mass of 144 and 36 kDa, respectively; the pH-activity optimum is 8.8. Rather than being stimulated, aldolase activity is inhibited to varying degrees by added divalent metal ions, whereas a number of metal ion-chelating agents have no effect. An absolute requirement for added thiol compounds could not be shown, but 2-mercaptoethanol enhances activity 2-fold, and added Hg2+ as well as p-mercuribenzoate or dithiodipyridine markedly inhibit catalysis. Incubation of the enzyme with either pyruvate or glyoxylate in the presence of NaBH4 causes extensive loss of aldolase activity concomitant with stable binding of approximately 1.0-1.5 mol of 14C-labeled substrate/mol of enzyme. The circular dichroism spectrum for native aldolase is characteristic of an alpha-helix; incubation of the enzyme with glyoxylate has no effect on this spectrum, but it is considerably altered by pyruvate. Bovine kidney aldolase shows no stereospecificity in catalyzing the aldol cleavage of the two optical isomers of 2-keto-4-hydroxyglutarate, and although it also catalyzes the beta-decarboxylation of oxalacetate, its decarboxylase/aldolase activity ratio is lower than that seen with the pure enzyme from either bovine liver or Escherichia coli.
Dekker
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Department of Biological Chemistry, The University of Michigan, Ann Arbor 48109-0606.
Kitson
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D010084:Oxidation-Reduction; D007652:Oxo-Acid-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism
D000596:Amino Acids; D013438:Sulfhydryl Compounds; D007652:Oxo-Acid-Lyases; C021415:4-hydroxy-2-oxoglutarate aldolase