How Enzymes Control the Reactivity of Adenosylcobalamin:  Effect on Coenzyme Binding and Catalysis of Mutations in the Conserved Histidine-Aspartate Pair of Glutamate Mutase
學年 85
學期 2
出版（發表）日期 1997-04-01
作品名稱 How Enzymes Control the Reactivity of Adenosylcobalamin:  Effect on Coenzyme Binding and Catalysis of Mutations in the Conserved Histidine-Aspartate Pair of Glutamate Mutase
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(25), pp.7884-7889
摘要 Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze unusual isomerizations that proceed through the formation of radical intermediates. It shares a structurally similar cobalamin-binding domain with methylcobalamin-dependent methionine synthase. In particular, both proteins contain the "DXHXXG" cobalamin-binding motif, in which the histidine provides the axial ligand to cobalt. The effects of mutating the conserved histidine and aspartate residues in methionine synthase have recently been described [Jarrett, J. T., Amaratunga, M., Drennan, C. L., Scholten, J. D., Sands, R. H., Ludwig, M. L., & Matthews, R. G. (1996) Biochemistry 35, 2464-2475]. Here, we describe how similar mutations in the "DXHXXG" motif of glutamate mutase affect coenzyme binding and catalysis in an adenosylcobalamin-dependent reaction. The mutations made in the MutS subunit of glutamate mutase were His16Gly, His16Gln, Asp14Asn, Asp14Glu, and Asp14Ala. All the mutations affect, in varying degrees, the rate of catalysis, the affinity of the protein for the coenzyme, and the coordination of cobalt. Mutations of either Asp14 or His16 decrease k(cat) by 1000-fold, and whereas cob(II)alamin accumulates as an intermediate in the wild-type enzyme, it does not accumulate in the mutants, suggesting the rate-determining step is altered. The apparent Kd for adenosylcobalamin is raised by about 50-fold when His16 is mutated and by 5-10-fold when Asp16 is mutated. There are extensive differences between the UV-visible spectra of wild-type and mutant holoenzymes, indicating that the mutant enzymes coordinate cobalt less well. Overall, the properties of these mutants differ quite markedly from those observed when similar mutations were introduced into methionine synthase.
關鍵字
語言 en
ISSN 0006-2960
期刊性質 國外
收錄於
產學合作
通訊作者
審稿制度 否
國別 USA
公開徵稿
出版型式 ,電子版,紙本
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