Kinetic and Mutational Studies of Three NifS Homologs from Escherichia coli. Mechanistic Difference between L-Cysteine Desulhrase and L-Selenocysteine Lyase Reactions.
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概要
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We have purified three NifS homologs from Escherichia coli, CSD, CsdB, and IscS, that appear to be involved in iron-sulfur cluster formation and/or the biosynthesis of selenophosphate. All three homologs catalyze the elimination of Se and S from L-selenocysteine and L-cysteine, respectively, to form L-alanine. These pyridoxal 5'-phosphate enzymes were inactivated by abortive transamination, yielding pyruvate and a pyridox-amine 5'-phosphate form of the enzyme. The enzymes showed non-Michaelis-Menten behavior for L-selenocysteine and L-cysteine. When pyruvate was added, they showed Michaelis-Menten behavior for L-selenocysteine but not for L-cysteine. Pyruvate signifi-cantly enhanced the activity of CSD toward L-selenocysteine. Surprisingly, the enzyme activity toward L-cysteine was not increased as much by pyruvate, suggesting the pres-ence of different rate-limiting steps or reaction mechanisms for L-cysteine desulfuriza-tion and the degradation of L-selenocysteine. We substituted Ala for each of Cys 358 in CSD, Cys 364 in CsdB, and Cys 328 in Iscs, residues that correspond to the catalytically essential Cys 325 of Azotobacter vinelandii NifS. The enzyme activity toward L-cysteine was almost completely abolished by the mutations, whereas the activity toward L-sele-nocysteine was much less affected. This indicates that the reaction mechanism of L-cys-teine desulfurization is different from that of L-selenocysteine decomposition, and that the conserved cysteine residues play a critical role only in L-cysteine desulfurization.
- 社団法人 日本生化学会の論文
著者
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Yoshimura Tohru
Institute For Chemical Research Kyoto University:(present Address)department Of Applied Molecular Bi
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Esaki Nobuyoshi
Institute For Chemical Resarch Kyoto University
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Kurihara Tatsuo
Institute For Chemical Research Kyoto University
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Mihara Hisaaki
Institute For Chemical Research Kyoto University
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