Hydrogen sulfide (H 2 S) has been observed in relatively high concentrations in the mammalian brain and has been shown to act as a neuromodulator. However, there is confusion in the literature regarding the actual source of H 2 S production. Reactions catalyzed by the cystathionine ␤-synthase enzyme (CBS) are one possible source for the production of H 2 S. Here we show that the CBS enzyme can efficiently produce H 2 S via a ␤-replacement reaction in which cysteine is condensed with homocysteine to form cystathionine and H 2 S. The production of H 2 S by this reaction is at least 50 times more efficient than that produced by hydrolysis of cysteine alone via ␤-elimination. Kinetic studies demonstrate that the K m and K cat for cysteine is 3-fold higher and 2-fold lower, respectively, than that for serine. Consistent with these data, in vitro reconstitution studies show that at physiologically relevant concentrations of serine, homocysteine, and cysteine, about 5% of the cystathionine formed is from cysteine. We also show that AdoMet stimulates this H 2 S producing reaction but that there is no evidence for stimulation by calcium and calmodulin as reported previously. In summary, these results confirm the ability of CBS to produce H 2 S, but show in contrast to prior reports that the major mechanism is via ␤-replacement and not cysteine hydrolysis. In addition, these studies provide a biochemical explanation for the previously inexplicable homocysteinelowering effects of N-acetylcysteine treatments in humans.Recently, there has been increased interest in endogenously produced hydrogen sulfide (H 2 S) as a physiologically important molecule. Relatively high concentrations of H 2 S have been observed in the brains of rats, humans, and cows (1-3). At physiological concentrations it has been shown that H 2 S enhances N-methyl-D-asparate receptor-mediated response and can modify long term potentiation (4 -6). H 2 S also inhibits smooth muscle cell proliferation via the mitogen-activated protein kinase pathway and protects neurons against oxidative stress (7). H 2 S also appears to have an effect on the cardiovascular system, acting as a vasorelaxant by increasing potassium-ATP channel currents (8). Taken together, these observations suggest that endogenously produced H 2 S is an important regulatory molecule in humans.How is endogenous H 2 S produced? Potential sources are alternative reactions catalyzed by the enzyme cystathionine ␤-synthase (CBS) 1 (6). The normal cellular function of CBS is to catalyze the condensation of serine with homocysteine to form cystathionine and water, a key reaction in the transsulfuration pathway. CBS uses pyridoxal phosphate (PLP) as a co-factor and is a member of the ␤-family or fold type II of PLP containing enzymes. Enzymes in this family characteristically have the ability to catalyze ␤-replacement and ␤-elimination reactions from a variety of different substrates (9).There are two potential mechanisms through which CBS could produce H 2 S. First, CBS could catalyze the production o...

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.

Our studies of cystathionine ␤-synthase from Saccharomyces cerevisiae (yeast) are aimed at (1) clarifying the cofactor dependence and catalytic mechanism and (2) 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 ␤-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 ␤-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 ␤-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.Elevated plasma homocysteine is an important risk factor in coronary heart disease and other human diseases (1-3). One of the two major routes for detoxication of homocysteine is the pyridoxal phosphate (PLP) 1 -dependent ␤-replacement reactionThe deduced sequences of human (4,5), rat (6), and Saccharomyces cerevisiae (yeast) (7,8) CBS are similar. The finding that human CBS complements the cysteine auxotrophy of a yeast strain lacking CBS (5) demonstrates the functional conservation of the human and yeast genes.The remarkable observation that the sequence of rat CBS (6) is identical to the sequence of rat hemoprotein H-450 (9) led to the discovery that rat and human CBS contain both PLP and heme (10). Heme may play a role in redox regulation of the human enzyme and in binding homocysteine (11,12). Although yeast CBS has been purified to homogeneity (13), the absorption spectrum and cofactor content have not been reported. Here, we demonstrate that purified yeast CBS contains PLP but not heme. Because the absence of heme facilitates spectroscopic studies of the PLP and of enzyme-substrate intermediates, we are able to demonstrate directly that CBS converts L-serine to an aminoacrylate intermediate, as expected for a PLP enzyme that catalyzes a ␤-replacement reaction (14,15). EXPERIMENTAL PROCEDURESChemicals-L-Cystathionine and L-serine were from Fluka. ␦-Aminolevulinic acid, L-homocysteine thiolactone, aprotinin, pepstatin A, leupeptin, benzamidine hydrochloride, TPCK, TLCK, and PMSF were from S...