Characteristics of L-alanine:4,5-dioxovaleric acid transaminase: an alternate pathway of heme biosynthesis in yeast.
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 degrees 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 X 10(-7), 5.0 X 10(-7), and 1.05 X 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.
Hoare
K
K
School of Environmental Sciences, Jawaharalal Nehru University, New Delhi, India.
Datta
K
K
D002176:Candida albicans / Q000201:enzymology*; D002845:Chromatography; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D003600:Cytosol / Q000201:enzymology; D017886:Durapatite; D006418:Heme / Q000096:biosynthesis*; D006882:Hydroxyapatites; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity; D000637:Transaminases / Q000302:isolation & purification / Q000378:metabolism*
D006882:Hydroxyapatites; D006418:Heme; D017886:Durapatite; D000637:Transaminases; C019687:aminolevulinate aminotransferase