Copurification of hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase of Saccharomyces cerevisiae: characterization of hydroxyethylthiazole kinase as a bifunctional enzyme in the thiamine biosynthetic pathway.
Mutants of Saccharomyces cerevisiae resistant to 2-amino-4-methyl-5-beta-hydroxyethylthiazole, an antimetabolite of 4-methyl-5-beta-hydroxyethylthiazole (hydroxyethylthiazole), which are deficient in the activities of both hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase, involved in the pathway of de novo synthesis of thiamine in S. cerevisiae, have been isolated. Genetic analysis revealed that the mutation occurs at a single gene in the nucleus. The two enzyme activities were copurified to apparent homogeneity, and the molecular masses of the purified proteins were found to be approximately 470 and 60 kDa, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. Hydroxyethylthiazole kinase was specific for ATP and Mg2+, although to a lesser extent a combination with other nucleoside triphosphates or divalent cations could replace them. p-Chloromercuribenzoate was a potent inhibitor of the enzyme, and the inhibition was prevented by the addition of 2-mercaptoethanol. These findings indicate that yeast hydroxyethylthiazole kinase is a bifunctional enzyme with thiamine-phosphate pyrophosphorylase activity, which is an octamer of identical 60-kDa subunits.
Kawasaki
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Department of Biochemistry, Kyoto Prefectural University of Medicine, Japan.
D019883:Alkyl and Aryl Transferases; D002413:Cations, Divalent / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D010770:Phosphotransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013831:Thiamine / Q000096:biosynthesis*; D013844:Thiazoles / Q000378:metabolism; D014166:Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D002413:Cations, Divalent; D009097:Multienzyme Complexes; D013844:Thiazoles; C051255:2-amino-4-methyl-5-hydroxyethylthiazole; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C435495:thiamin phosphate synthase; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C051254:hydroxyethylthiazole kinase; D013831:Thiamine