教師資料查詢 | 類別: 期刊論文 | 教師: 陳灝平 HAO-PING CHEN (瀏覽個人網頁)

標題:Adenosylcobalamin-Dependent Glutamate Mutase: Examination of Substrate and Coenzyme Binding in an Engineered Fusion Protein Possessing Simplified Subunit Structure and Kinetic Properties
學年86
學期1
出版(發表)日期1997/12/01
作品名稱Adenosylcobalamin-Dependent Glutamate Mutase: Examination of Substrate and Coenzyme Binding in an Engineered Fusion Protein Possessing Simplified Subunit Structure and Kinetic Properties
作品名稱(其他語言)
著者Chen, H.P.; Marsh, EN.
單位淡江大學化學學系
出版者Washington, DC: American Chemical Society
著錄名稱、卷期、頁數Biochemistry 36(48), pp.14939-14945
摘要Glutamate mutase is comprised of two weakly associating subunits; E and S, that combine to form the coenzyme binding site. The active holoenzyme assembles in a kinetically complex process in which both the stoichiometry and apparent Kd for adenosylcobalamin (AdoCbl) are dependent upon the relative concentrations of the two subunits, as is the enzyme's specific activity. To facilitate mechanistic and structural studies on this enzyme we have genetically fused the S subunit to the C-terminus of the E subunit through an 11 amino acid (Gly-Gln)5-Gly linker segment. This protein, GlmES, binds AdoCbl stoichiometrically and neither the affinity for AdoCbl nor the turnover number depends upon protein concentration. The kcat and Km for both substrate and coenzyme, together with the deuterium isotope effects on Vmax and Vmax/Km, have been determined for the GlmES-catalyzed reaction proceeding in both directions. Compared with wild type, the affinity for AdoCbl is unchanged, but for the conversion of L-glutamate to (2S,3S)-3-methylaspartate both kcat and Km for L-glutamate are decreased by about a third and the isotope effects are reduced, suggesting product release to be more rate-limiting. To test hypotheses concerning the activation of the coenzyme, we examined the binding of adenosylcobalamin, methylcobalamin, and cob(II)alamin to the enzyme. Each of these is bound with essentially the same affinity (2 microM), suggesting that, contrary to expectations, interactions between the protein and the adenosyl moiety do not serve to weaken the cobalt-carbon bond in the ground state.
關鍵字
語言英文
ISSN0006-2960
期刊性質國外
收錄於
產學合作
通訊作者Marsh, EN.
審稿制度
國別美國
公開徵稿
出版型式紙本
相關連結
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