• BMB Reports
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• v.29 no.3
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• pp.210-214
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• 1996

The pH dependence of the kinetic parameters of mutant O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium LT-2 has been determined in order to obtain information on the chemical mechanism. The initial velocity pattern obtained by varying the concentrations of OAS at several fixed concentrations of TNB, shows an intersection on the left of the ordinate at pH 7.0, indicating that the kinetic mechanism is a sequential mechanism in which substrate inhibition by OAS is observed while the wild type enzyme showed a ping pong mechanism. The values of $V/E_t$, $V/K_{OAS}E_{t}$ and $V/K_{TNB}E_{t}$ decreased by about 68%, 14% and 16% as compared with the wild type enzyme. The $V/K_{OAS}E_{t}$ is a pK of 6.5 on the acid side of the pH profile, and the $V/K_{TNB}$ is pH independent. As compared with the wild type enzyme, the pKs in the V/K profiles are shifted, reflecting that binding of the cofactor in free E:OAS is less asymmetric.

• BMB Reports
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• v.29 no.1
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• pp.32-37
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• 1996

The cysteine 43, potentially important in the activity of O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium, has been changed to serine. This mutant enzyme (C43S) has been studied in order to gain insight into the structural basis for the binding of inhibitor, substrate and product. UV-visible spectra of C43S exhibit the same spectral change in the presence of OAS as that observed with wild type enzyme, indicating C43S will form an ${\alpha}$-aminoacrylate Schiff base intermediate. At pH 6.5, however, the deacetylase activity of C43S is much higher than wild type enzyme indicating that cysteine 43 plays a role in stabilizing the ${\alpha}$-aminoacrylate intermediate. The fluoroscence spectrum of C43S exhibits a ratio of emission at 340 to 502 nm of 16.9, reflecting the lower fluorescence of PLP and indicating that the orientation of cofactor and tryptophan are different from that of the wild type enzyme. The emission spectrum of C43S in the presence of OAS gives two maxima at 340 and 535 nm. The 535 nm emission is attributed to the fluoroscence of the ${\alpha}$-aminoacrylate intermediate. The visible circular dichroic spectrum was similar to wild type enzyme, but the negative effect observed at 530~550 nm and the molar ellipicity values for the mutant are decreased by about 50% compared to wild type enzyme. The circular dichroic and fluoroscence studies suggest binding of the cofactor is less asymmetric in C43S than in the wild type enzyme.

• KSBB Journal
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• v.23 no.5
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• pp.425-430
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• 2008

Hydrogen sulfide ($H_2S$) is a by-product of metabolism of amino acids including sulfur and alcoholic fermentation, it is generally thought of in terms of a poisonous gas. Though $H_2S$ can have a negative impact on the perceived quality of fermented drinks due to an undesirable aroma, it plays prominent roles as a neuromodulator in the mammalian brain as well as a smooth muscle relaxant. Nowadays studies on the proteins which produce $H_2S$ are carried out in various fields such as structure, function, and metabolism. Here we propose to develop a simple and rapid $H_2S$ forming assay method, which will lead to speed up preparing the $H_2S$ forming proteins for identification by MALDI-TOF MS analysis. We detected three kinds of proteins which produce $H_2S$ in the crude extract of Saccharomyces cerevisiae. Those proteins were cystathionie $\beta$-synthase, O-acetylserine sulfhydrylase, and cystathionine $\gamma$-lyase.