Yeast cystathionine beta-synthase is a pyridoxal phosphate enzyme but, unlike the human enzyme, is not a heme protein.
Our studies of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are aimed at clarifying the cofactor dependence and catalytic mechanism and obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease). We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine beta-synthase. The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine beta-synthase. The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism. The reaction of the enzyme with L-serine in the absence of L-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm. The formation of this intermediate from the product, L-cystathionine, demonstrates the partial reversibility of the reaction. Our results establish the overall catalytic mechanism of yeast cystathionine beta-synthase and provide a useful system for future studies of structure and function. The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymes O-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase.
Jhee
K H
KH
Laboratory of Biochemistry and Genetics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0830, USA.
McPhie
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P
Miles
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EW
D000818:Animals; D002384:Catalysis; D002942:Circular Dichroism; D003541:Cystathionine beta-Synthase / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000032:analysis; D006801:Humans; D008956:Models, Chemical; D011487:Protein Conformation; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012694:Serine / Q000378:metabolism; D013054:Spectrophotometry, Atomic
D006418:Heme; D012694:Serine; D003541:Cystathionine beta-Synthase