Domain architecture of the heme-independent yeast cystathionine beta-synthase provides insights into mechanisms of catalysis and regulation.
Cystathionine beta-synthase from yeast (Saccharomyces cerevisiae) provides a model system for understanding some of the effects of disease-causing mutations in the human enzyme. The mutations, which lead to accumulation of L-homocysteine, are linked to homocystinuria and cardiovascular diseases. Here we characterize the domain architecture of the heme-independent yeast cystathionine beta-synthase. Our finding that the homogeneous recombinant truncated enzyme (residues 1-353) is catalytically active and binds pyridoxal phosphate stoichiometrically establishes that the N-terminal residues 1-353 compose a catalytic domain. Removal of the C-terminal residues 354-507 increases the specific activity and alters the steady-state kinetic parameters including the K(d) for pyridoxal phosphate, suggesting that the C-terminal residues 354-507 compose a regulatory domain. The yeast enzyme, unlike the human enzyme, is not activated by S-adenosyl-L-methionine. The truncated yeast enzyme is a dimer, whereas the full-length enzyme is a mixture of tetramer and octamer, suggesting that the C-terminal domain plays a role in the interaction of the subunits to form higher oligomeric structures. The N-terminal catalytic domain is more stable and less prone to aggregate than full-length enzyme and is thus potentially more suitable for structure determination by X-ray crystallography. Comparisons of the yeast and human enzymes reveal significant differences in catalytic and regulatory properties.
Jhee
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Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 8, Room 225, 8 Center Drive, MSC 0830, Bethesda, Maryland, USA.
McPhie
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Miles
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D001483:Base Sequence; D002384:Catalysis; D003541:Cystathionine beta-Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013045:Species Specificity
D017931:DNA Primers; D003541:Cystathionine beta-Synthase