title: Purification and characterization of 2-keto-3-deoxy-6-phosphogluconate aldolase from Azotobacter vinelandii: evidence that the enzyme is bifunctional towards 2-keto-4-hydroxy glutarate cleavage.
abstract: 2-keto-3-deoxy-6-phosphogluconate aldolase (E.C. 4.1.2.14) has been purified in two chromatographic steps to 99% purity in 73% overall yield from Azotobacter vinelandii. The pure enzyme is a 70 kD trimeric Class I aldolase, inhibitable by bromopyruvate or pyruvate plus sodium borohydride, with a specific activity of 625 mumol per min per mg protein and a Km of 38 microM for 2-keto-3-deoxy-6-phosphogluconate. The enzyme also has 2-keto-4-hydroxy glutarate aldolase (E.C. 4.1.3.16) activity, with a specific activity of 4.8 mumol per min per mg protein and a Km of 39 microM. 2-keto-4-hydroxy glutarate inhibits the 2-keto-3-deoxy-6-phosphogluconate aldolase activity of the enzyme with an apparent Ki of 0.17 mM. Slow steps following formation of the Schiff base intermediate between KHG and the enzyme are responsible for both the slower turnover of this substrate and for its inhibitory effect.
authors.affiliation: Department of Biochemistry, Michigan State University, East Lansing 48824.
authors.forename: T S
authors.initials: TS
authors.lastname: Taha
authors.forename: T L
authors.initials: TL
authors.lastname: Deits
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D016948:Azotobacter vinelandii / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007656:Ketoglutaric Acids / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011487:Protein Conformation; D012492:Salts / Q000494:pharmacology
chemical_list: D007656:Ketoglutaric Acids; D012492:Salts; C018989:2-keto-4-hydroxyglutarate; D000446:Aldehyde-Lyases; C100131:phospho-2-keto-3-deoxy-gluconate aldolase