Purification of δ‐aminolevulinate dehydratase from genetically engineered yeast

Saccharomyces cerevisiae transformed with a multicopy plasmid carrying the yeast structural gene HEM2 , which codes for δ‐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 μmol h −1 per mg protein at pH 9·4 and 37·5°C. In most respects the yeast enzyme resembles mammalian enzymes. It is a homo‐octamer with an apparent M r , of 275 000, as determined by centrifugation in glycerol density gradients, and under denaturing conditions behaved as a single subunit of M r ≃ 37 000. The enzyme requires reduced thiol compounds to maintain full activity, and maximum activity was obtained in the presence of 1·0 m M ‐Zn 2+ . It is sensitive to inhibition by the heavy metal ions Pb 2+ and Cu 2+ . The enzyme exhibits Michaelis‐Menten kinetics and has an apparent K m of 0·359 m M . Like dehydratases from animal tissues, the yeast enzyme is rather thermostable. During the purification process an enhancement in total δ‐aminolevulinate dehydratase activity suggested the possibility that removal of an inhibitor of the enzyme could be occurring.