Substrate Inhibition of<scp>L</scp>-Cysteine α,β-Elimination Reaction Catalyzed by<scp>L</scp>-Cystathionine γ-Lyase of<i>Saccharomyces cerevisiae</i>

Yamagata, Shuzo; Isaji, Motomi; Yamane, Tomoko; Iwama, Tetsuo

doi:10.1271/bbb.66.2706

Cited by 14 publications

(24 citation statements)

References 25 publications

(34 reference statements)

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“…l -cys can enter into the active site of the enzyme, resulting in the formation of cysteine-PLP external aldimine, which next undergoes intramolecular cyclization to form a thiazolidine ring between the formyl group of PLP and sulfhydryl groups of l -cys. We suggest that the 335 nm peak could represent the formation of a thiazolidine adduct, as already observed for human and yeast CGL [ 18 , 19 ] and for other PLP-dependent enzymes [ 20 ]. This finding may offer an explanation for the inhibitory effect of l -cys.…”

Section: Resultssupporting

confidence: 75%

See 1 more Smart Citation

Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii

Maresi

Janson

Fruncillo

et al. 2018

IJMS

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Sulfur-containing amino acids play essential roles in many organisms. The protozoan parasite Toxoplasma gondii includes the genes for cystathionine β-synthase and cystathionine γ-lyase (TgCGL), as well as for cysteine synthase, which are crucial enzymes of the transsulfuration and de novo pathways for cysteine biosynthesis, respectively. These enzymes are specifically expressed in the oocyst stage of T. gondii. However, their functionality has not been investigated. Herein, we expressed and characterized the putative CGL from T. gondii. Recombinant TgCGL almost exclusively catalyses the α,γ-hydrolysis of l-cystathionine to form l-cysteine and displays marginal reactivity toward l-cysteine. Structure-guided homology modelling revealed two striking amino acid differences between the human and parasite CGL active-sites (Glu59 and Ser340 in human to Ser77 and Asn360 in toxoplasma). Mutation of Asn360 to Ser demonstrated the importance of this residue in modulating the specificity for the catalysis of α,β- versus α,γ-elimination of l-cystathionine. Replacement of Ser77 by Glu completely abolished activity towards l-cystathionine. Our results suggest that CGL is an important functional enzyme in T. gondii, likely implying that the reverse transsulfuration pathway is operative in the parasite; we also probed the roles of active-site architecture and substrate binding conformations as determinants of reaction specificity in transsulfuration enzymes.

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Section: Resultssupporting

confidence: 75%

“…Notably, after dialysis of the enzyme thiazolidine complex, the 335 nm peak decreased and the 421 nm peak was regenerated, indicating that thiazolidine formation is reversible ( Figure 4 B, inset). Reversibility of thiazolidine formation has been reported previously [ 18 , 20 , 21 , 23 , 24 , 25 ].…”

Section: Resultsmentioning

confidence: 76%

Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii

Maresi

Janson

Fruncillo

et al. 2018

IJMS

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“…We did not observe any evidence of significant H 2 S formation in yeast extracts using our gel overlay assay when only cysteine was added, even though yeast has significant levels of CGL activity (24). Furthermore, we failed to observe any detectable H 2 S formation in the extracts of mouse livers of animals deleted for endogenous CBS.…”

Section: Discussioncontrasting

confidence: 56%

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

Chen

Jhee

Kruger

2004

Journal of Biological Chemistry

304

214

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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...

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“…According to Yamagata et al, in addition to binding to the enzyme, Lcysteine may also bind to the exogenous PLP cofactor to form thiazolidines. 26 This may offer an explanation for the subsequent decrease in reaction rates beyond an optimum concentration of exogenous PLP (approximately 50 μM; Fig. 6a).…”

Section: Catalysis Of H 2 S Production From Wild-type Cse In the Presmentioning

confidence: 94%

Site-Directed Mutagenesis on Human Cystathionine-γ-Lyase Reveals Insights into the Modulation of H2S Production

Huang

Chua

Yew

et al. 2010

Journal of Molecular Biology

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Substrate Inhibition ofL-Cysteine α,β-Elimination Reaction Catalyzed byL-Cystathionine γ-Lyase ofSaccharomyces cerevisiae

Cited by 14 publications

References 25 publications

Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii

Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii

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

Site-Directed Mutagenesis on Human Cystathionine-γ-Lyase Reveals Insights into the Modulation of H2S Production

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