Characteristics of L-alanine: 4,5-dioxovaleric acid transaminase: an alternate pathway of heme biosynthesis in yeast

Hoare, Kishalay ; Datta, Kasturi (1990) Characteristics of L-alanine: 4,5-dioxovaleric acid transaminase: an alternate pathway of heme biosynthesis in yeast Archives of Biochemistry and Biophysics, 277 (1). pp. 122-129. ISSN 0003-9861

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Official URL: http://dx.doi.org/10.1016/0003-9861(90)90559-H

Related URL: http://dx.doi.org/10.1016/0003-9861(90)90559-H

Abstract

The present study reports for the first time the presence of the enzyme L-alanine:4,5-dioxovaleric acid transaminase (EC 2.6.1.43), which catalyzes the irreversible synthesis of 5-aminolevulinic acid in three strains of yeast: Candida albicans 3100, Saccharomyces cerevisiae 3059, and S. cerevisiae S288C. The enzyme was predominantly present in the cytosol and was purified from C. albicans 3100 by about 200-fold with an overall yield of 23% to apparent homogeneity. The purification procedure involved heat treatment, followed by affinity chromatography on L-alanine-Sepharose CL-4B, ion-exchange chromatography on DEAE-cellulose DE-52, and chromatography on hydroxyapatite. As judged by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appeared to be a monomeric protein of relative molecular mass 59,000. The enzyme activity was stimulated by pyridoxal phosphate and was optimally active at 60 °C. The purified enzyme had an isoelectric point of 4.7 and a pH optimum of 7.2. Km values of the enzyme for L-alanine, pyridoxal phosphate, and 4,5-dioxovaleric acid were 2.8 × 10-7, 5.0 × 10-7, and 1.05 × 10-5 M, respectively. The Vmax of the enzymatic reaction was 5 x 10-6 mol/mg/h. Antibody raised against the purified yeast L-alanine:4,5-dioxovaleric acid transaminase did not cross-react with the same enzyme from rat liver and kidney.

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