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• 作者：Xuemei M. H. Chen ; Olivier Ploux ; and Hung-wen Liu
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：December 24, 1996
• 年：1996
• 卷：35
• 期：51
• 页码：16412 - 16420
• 全文大小：490K
• 年卷期：v.35,no.51(December 24, 1996)
• ISSN：1520-4995

文摘

CDP-6-deoxy-L-threo-D-glycero-4-hexulose3-dehydrase (E₁), together with its reductase(E₃),catalyzes a novel deoxygenation reaction essential for the biosynthesisof 3,6-dideoxyhexoses. In anattempt to gain evidence substantiating theE₁·E₃ complex formation as a prerequisite forthe C-3deoxygenation activity, we have carried out experiments to study theinteraction between these two proteins.The detection of a new species when a mixture of E₁and E₃ was analyzed by size-exclusionchromatographywas the initial indication supporting the proposed complex formation.Additional evidence for the expectedcomplex formation was provided by the change of the CD spectrum ofE₁ upon its coupling with E₃.Thefact that the catalytic efficiency of this system is limited by thequantity of one enzyme, which becomescatalytically competent only after coupling with the second enzyme,further illustrated the importance ofsuch a complex formation to the deoxygenation activity. By usingthe two-hybrid system which scoresfor interactions between two proteins coexpressed in yeast, theE₁·E₃ complex formation in vivowas alsofirmly established. These results, when considered with theincompatibility of other electron transferproteins as replacements for E₃ in this electron relay,nicely demonstrated the specificity of theE₁-E₃recognition. The apparent dissociation constant of theE₁·E₃ complex formed in rapid equilibriumwasestimated to be 288 ± 22 nM from the correlation between the initialrate of the overall reaction and theconcentration of one protein component, and the stoichiometry betweenE₃ and E₁ of this complex wasdeduced as 1.7. Interestingly, while the conformation of theE₁·E₃ complex was sensitive to thesaltconcentration in the buffer, the decrease in the catalytic activity athigh ionic strength was most likelydue to the retardation of the electron transfer mediated byE₃. In conjunction with early mechanisticstudies, the present data establish the significance of theE₁·E₃ complex formation for catalysisand,consequently, corroborate the mechanism proposed for the overalldeoxygenation process.