1. Purification of δ-aminolevulinate dehydratase from genetically engineered yeast
- Author
-
Leda M. Borralho, Anita D. Panek, James R. Mattoon, and Claudio H. D. Ortiz
- Subjects
Porphobilinogen ,Saccharomyces cerevisiae ,Bioengineering ,Applied Microbiology and Biotechnology ,Biochemistry ,Cell-free system ,Transformation, Genetic ,Genetics ,Protamines ,Sulfhydryl Compounds ,Enzyme inducer ,chemistry.chemical_classification ,biology ,Structural gene ,Temperature ,Porphobilinogen Synthase ,Hydrogen-Ion Concentration ,biology.organism_classification ,Yeast ,Enzyme assay ,Kinetics ,Zinc ,Enzyme ,chemistry ,Dehydratase ,biology.protein ,Plasmids ,Biotechnology - Abstract
Saccharomyces cerevisiae transformed with a multicopy plasmid carrying the yeast structural gene HEM2, which codes for delta-aminolevulinate dehydratase, was enriched 20-fold in the enzyme. Beginning with cell-free extracts of transformed cells, the dehydratase was purified 193-fold to near-homogeneity. This represents a 3900-fold purification relative to the enzyme activity in normal, untransformed yeast cells. The specific activity of the purified enzyme was 16.2 mumol h-1 per mg protein at pH 9.4 and 37.5 degrees C. In most respects the yeast enzyme resembles mammalian enzymes. It is a homo-octamer with an apparent Mr of 275,000, as determined by centrifugation in glycerol density gradients, and under denaturing conditions behaved as a single subunit of Mr congruent to 37,000. The enzyme requires reduced thiol compounds to maintain full activity, and maximum activity was obtained in the presence of 1.0 mM-Zn2+. It is sensitive to inhibition by the heavy metal ions Pb2+ and Cu2+. The enzyme exhibits Michaelis-Menten kinetics and has an apparent Km of 0.359 mM. Like dehydratases from animal tissues, the yeast enzyme is rather thermostable. During the purification process an enhancement in total delta-aminolevulinate dehydratase activity suggested the possibility that removal of an inhibitor of the enzyme could be occurring.
- Published
- 1990