Domain Architecture of the Heme-Independent Yeast Cystathionine β-Synthase Provides Insights into Mechanisms of Catalysis and Regulation

Self Cite

300

214

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...

Section: Cbs Expressionmentioning

confidence: 99%

“…Using this mechanism CBS can produce H 2 S from the reaction of L-cysteine and 2-mercaptoethanol to form S-hydroxyethyl-L-cysteine and H 2 S (Fig. 1, Alternate Reaction 2) (11). While this reaction would not be expected to occur in vivo, a similar ␤-replacement reaction could occur by the condensation of homocysteine with cysteine ( Fig.…”

mentioning

confidence: 99%

Production of the Neuromodulator H2S by Cystathionine β-Synthase via the Condensation of Cysteine and Homocysteine

Chen

Jhee

Kruger

2004

Self Cite

300

214

“…AdoMet plays an important role in the regulation of these two competing pathways by inhibiting MTHFR (6 -8). AdoMet also activates cystathionine ␤-synthase (CBS) in mammalian cells (9), but this regulation is absent in yeast (10,11). However, AdoMet is required for full cysteine-mediated transcriptional regulation of sulfur assimilation genes (5).…”

mentioning

confidence: 99%

Regulation of S-Adenosylmethionine Levels in Saccharomyces cerevisiae

Chan¹,

Appling

2003

Folate derivatives, which are present in virtually every known organism and cell type, mediate one-carbon (C 1 ) transfers that play essential roles in several key cellular processes. Folate-dependent one-carbon metabolism is necessary for the syntheses of purines, thymidylate, formylmethionyl-tRNA, glycine, serine, and methionine (1, 2). Furthermore, many methylated molecules such as nucleic acids, proteins, and lipids are formed by transmethylation reactions with S-adenosylmethionine. This latter compound is synthesized indirectly by a folate-dependent pathway (Fig. 1) and is second only to ATP as the most abundantly used cofactor in metabolic reactions. In addition to serving as a methyl group donor, the propylamine group of decarboxylated S-adenosylmethionine can be used in the synthesis of spermidine from putrescine; donation of a second propylamine group results in the formation of spermine.Methylenetetrahydrofolate reductase (MTHFR) 1 participates in methionine metabolism as shown in Fig.

“…4b). This band is attributed to the ketoenamine tautomer of the ␣-aminoacrylate absorbing at 450 -470 nm, as observed in O-acetylserine sulfhydrylase (22) and cystathionine ␤-synthase (23,24). This result indicates a negligible influence of IAG and GP on the equilibrium distribution of ␤-intermediates.…”

Section: Resultsmentioning

confidence: 69%

Allosteric Communication of Tryptophan Synthase

Biase

Tramonti

Bettati

et al. 2001

The ␣ 2 ␤ 2 tryptophan synthase complex is a model enzyme for understanding allosteric regulation. We report the functional and regulatory properties of the ␤S178P mutant. Ser-178 is located at the end of helix 6 of the ␤ subunit, belonging to the domain involved in intersubunit signaling. The carbonyl group of ␤Ser-178 is hydrogen bonded to Gly-181 of loop 6 of the ␣ subunit only when ␣ subunit ligands are bound. An analysis by molecular modeling of the structural effects caused by the ␤S178P mutation suggests that the hydrogen bond involving ␣Gly-181 is disrupted as a result of localized structural perturbations. The ratio of ␣ to ␤ subunit concentrations was calculated to be 0.7, as for the wild type, indicating the maintenance of a tight ␣؊␤ complex. Both the activity of the ␣ subunit and the inhibitory effect of the ␣ subunit ligands indole-3-acetylglycine and D,L-␣-glycerol-3-phosphate were found to be the same for the mutant and wild type enzyme, whereas the ␤ subunit activity of the mutant exhibited a 2-fold decrease. In striking contrast to that observed for the wild type, the allosteric effectors indole-3-acetylglycine and D,L-␣-glycerol-3-phosphate do not affect the ␤ activity. Accordingly, the distribution of L-serine intermediates at the ␤-site, dominated by the ␣-aminoacrylate, is only slightly influenced by ␣ subunit ligands. Binding of sodium ions is weaker in the mutant than in the wild type and leads to a limited increase of the amount of the external aldimine intermediate, even at high pH, whereas binding of cesium ions exhibits the same affinity and effects as in the wild type, leading to an increase of the ␣-aminoacrylate tautomer absorbing at 450 nm. Crystals of the ␤S178P mutant were grown, and their functional and regulatory properties were investigated by polarized absorption microspectrophotometry. These findings indicate that (i) the reciprocal activation of the ␣ and ␤ activity in the ␣2␤2 complex with respect to the isolated subunits results from interactions that involve residues different from ␤Ser-178 and (ii) ␤Ser-178 is a critical residue in ligand-triggered signals between ␣ and ␤ active sites.Allosteric proteins are key elements of cellular regulation, because they allow for a fine adaptation of the living organisms to changes in metabolite concentration and physico-chemical conditions. Tryptophan synthase is an allosteric enzyme, composed of two ␣ and two ␤ subunits, catalyzing the last two steps in the biosynthesis of L-tryptophan (1-3) (Fig. 1). The ␣ active site cleaves indole-3-glycerol phosphate into indole and D-glyceraldehyde-3-phosphate. The ␤ active site contains a pyridoxal 5Ј-phosphate molecule bound to Lys-87 via a Schiff base and catalyzes the replacement of the ␤-hydroxyl of L-serine with indole. The latter compound is intramolecularly channeled from the ␣ site via a hydrophobic tunnel connecting the ␣ and ␤ active sites, as proposed on the basis of the first crystal structure of the enzyme (4). The ␣ and ␤ activities are reciprocally modulated. Extensive functi...