Characterization of the S272A,D Site-Directed Mutations of O-Acetylserine Sulfhydrylase: Involvement of the Pyridine Ring in the 
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- 作者：Sebastian Daum ; Chia-Hui Tai ; and Paul F. Cook
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：January 14, 2003
- 年：2003
- 卷：42
- 期：1
- 页码：106 - 113
- 全文大小：152K
- 年卷期：v.42,no.1(January 14, 2003)
- ISSN：1520-4995
文摘
O-Acetylserine sulfhydrylase catalyzes the synthesis of L-cysteine from O-acetyl-L-serine (OAS)and inorganic bisulfide. An anti-E2 mechanism has been proposed for the OASS-catalyzed elimination ofacetate from OAS (Tai, C.-H., and Cook, P. F. (2001) Acc. Chem. Res. 34, 49-59). Site-directedmutagenesis was used to change S272 to alanine, which would be expected to eliminate the hydrogenbond to N1 of PLP or to aspartate, which would be expected to enhance the hydrogen-bonding interaction.Both mutant enzymes catalyze the overall reaction and are in fact still good enzymes, consistent with theproposed anti-E2 mechanism. Data suggest that hydrogen bonding to the pyridine ring does not play asignificant role in the 
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-elimination reaction catalyzed by OASS-A. The V/KOAS, which reflects thefirst half-reaction, is identical to the wild-type enzyme in the case of the S272D mutant enzyme and isdecreased by only a factor of 3 in the case of the S272A mutant enzyme. In the case of the alaninemutation, and to a lesser extent the aspartate mutation, a decrease in the rate of the elimination iscompensated by an increase in affinity for OAS, leading to the observed second-order rate constant, V/K.The decrease in the rate of the elimination is proposed to result from a change in the orientation of thebound cofactor, as might be expected since one of the ligands that determines the position of the boundPLP has been changed. Consistent with a change in the orientation of the cofactor are the results from anumber of the spectral probes. The visible CD data for the internal Schiff base have a molar ellipticity50% that of wild-type enzyme, and the -aminoacrylate intermediate has a molar ellipticity 25% that ofwild-type enzyme. The -aminoacrylate intermediate can be formed from L-cysteine and L-serine withthe S272A,D mutant enzymes, but not with the wild-type enzyme, and taken together with the increasedKd for the serine external Schiff base is consistent with a change in cofactor orientation in the active site.The long wavelength fluorescence emission for the S272A mutant enzyme, attributed to Förster resonanceenergy transfer (McClure, G. D., Jr., and Cook, P. F. (1994) Biochemistry 33, 1647-1683) has an intensitynear zero, as compared to significant fluorescence for the wild-type enzyme.
,-elimination reaction catalyzed by OASS-A. The V/KOAS, which reflects thefirst half-reaction, is identical to the wild-type enzyme in the case of the S272D mutant enzyme and isdecreased by only a factor of 3 in the case of the S272A mutant enzyme. In the case of the alaninemutation, and to a lesser extent the aspartate mutation, a decrease in the rate of the elimination iscompensated by an increase in affinity for OAS, leading to the observed second-order rate constant, V/K.The decrease in the rate of the elimination is proposed to result from a change in the orientation of thebound cofactor, as might be expected since one of the ligands that determines the position of the boundPLP has been changed. Consistent with a change in the orientation of the cofactor are the results from anumber of the spectral probes. The visible CD data for the internal Schiff base have a molar ellipticity50% that of wild-type enzyme, and the -aminoacrylate intermediate has a molar ellipticity 25% that ofwild-type enzyme. The -aminoacrylate intermediate can be formed from L-cysteine and L-serine withthe S272A,D mutant enzymes, but not with the wild-type enzyme, and taken together with the increasedKd for the serine external Schiff base is consistent with a change in cofactor orientation in the active site.The long wavelength fluorescence emission for the S272A mutant enzyme, attributed to Förster resonanceenergy transfer (McClure, G. D., Jr., and Cook, P. F. (1994) Biochemistry 33, 1647-1683) has an intensitynear zero, as compared to significant fluorescence for the wild-type enzyme.