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Abstract
L-Alanine:4,5-dioxovalerate aminotransferase, the enzyme that catalyzes the transamination between alanine and 4,5-dioxovalerate to yield delta-aminolevulinate and pyruvate, has been purified from extracts Clostridium tetanomorphum by acetone precipitation and successive tetanomorphum by acetone precipitation and successive chromatography on Sephadex G-150, hydroxyapatite, Octyl-Sepharose, and SP-Sephadex C-50. The enzyme is pure by the criterion of disc gel electrophoresis with varying polyacrylamide concentrations. It is dimeric, and has an apparent molecular weight of 111,000. Each molecule contains 2 molecules of pyridoxal 5-phosphate. The apparent Km values for 4,5-dioxovalerate and L-alanine are 0.26 and 1.96 mM, respectively. In addition to alanine, glutamate also is an effective amino group donor. The enzyme is inhibited by various keto acids as well as by inhibitors of pyridoxal phosphate-containing enzymes. It was possible to show that 4,5-dioxovalerate is formed by cultures of C. tetanomorphum when grown in the presence of 0.2 M levulinate, an inhibitor of 5-aminolevulinate dehydratase.
Highlights
~-Alanine:4,5-dioxovalerateaminotransferase, the organisms that contain the aminotransferase [8]
Studies with enzyme that catalyzes the transamination between al- Scenedesmus have indicated 4,5-dioxovalerate to be an interanine and 4,5-dioxovalerate t o yield 6-aminolevulinate mediate in the biosynthesis ofALA [9]
Substrate Specificity-All of the common amino acids were tested as amino group donors in the enzyme-catalyzed transamination
Summary
M levulinate, an inhibitor of 5-aminolevulinate dehydratase. Materials Chemicalswere obtained from the following sources:yeast extract, Difco;3,5-dibromolevulinic acid, Porphyrin Products, Logan, U T ethylacetoacetate, Aldrich; hydroxyapatite (Fast Flow), Calbiochem; Shortly after the discovery of the committed tetrapyrrole precursor, ALA' (I), extracts of avian erythrocytes [2] and of the photosynthetic bacterium Rhodopseudomonas spheroides [3] were shown to form this compound from succinyland Blue Dextran, Pharmacia. Molecular Weight Determination-The molecular weight of the aminotransferase was determined by molecular exclusion chromatography using a Sephadex G-150 column (1.6 X 29 cm) that had been equiiibrated with 50 m~ potassium phosphate buffer, pH 6.9, and calibrated with known standards These included Blue Dextran 2000 ( M , > l,000,OOO), catalase (M, = 230,000),bovine serum albumin ( M , = 68,000). Purity-Elution from the SP-Sephadex C-25 column pro- was attempted toshow the disappearanceof ALA by carrying duced a single, symmetrical protein peak with constant spe- out the enzyme incubation in 50 mM potassium phosphate, cific activity. On dodecyl sodiumsulfate-polyacrylamide gel rate of formation of ALA is constant for up to 1 h It was electrophoresis,a single bandappeared with anapparent shown by the methods used by Varticovski et al [13] that molecular weight of 55,500 (Fig. 4).
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