Purification of delta-aminolevulinate dehydratase from genetically engineered yeast.
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.
Departamento de Bioquímica, Universidade Federal do Rio de Janeiro, Cidade Universitária, Brazil.
L M
LM
Borralho
C H
CH
Ortiz
A D
AD
Panek
J R
JR
Mattoon
D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010957:Plasmids; D011162:Porphobilinogen / Q000378:metabolism; D000623:Porphobilinogen Synthase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011479:Protamines; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013438:Sulfhydryl Compounds; D013696:Temperature; D014170:Transformation, Genetic; D015032:Zinc / Q000494:pharmacology
D011479:Protamines; D013438:Sulfhydryl Compounds; D011162:Porphobilinogen; D000623:Porphobilinogen Synthase; D015032:Zinc