表达、纯化和表征杀稻瘟菌素的脱氨酶(BSD)曲霉菌terreus:催化锌酶结构的作用。
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木村M, Sekido年代,Isogai Y,我山口
表达、纯化和表征杀稻瘟菌素的脱氨酶(BSD)曲霉菌terreus:催化锌酶结构的作用。
,2000年6月,127 (6):955 - 63。
- PubMed ID
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10833262 (在PubMed]
- 文摘
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我们建立了一个有效的杀稻瘟菌素overproduction-purification系统S脱氨酶(BSD)从曲霉terreus利用cDNA克隆。纯化酶的分子质量估计表明BSD四聚物。这四聚物的形式非常耐变性SDS和SDS - page显示heat-modifiable行为;即。,BSD迁移慢得多(36 kDa的作为一个乐队)在其活性构象比完全变性多肽(kDa 13日)之前如果没有执行热处理在2% SDS电泳。从催化zinc-coordinating序列的存在预测主题保存在胞嘧啶核苷/核苷酸脱氨酶家族,BSD还包含一个锌/脱氨酶亚基。然而,预测催化功能出现不唯一的锌酶的作用。首先,滴定zinc-chelating sh组p-hydroxymercuriphenylsulfonate导致离解的BSD四聚物为不稳定的单体或二聚体。第二,消耗锌化学变性BSD的调整(guanidine-HCl或酸性pH值)导致不正确折叠的多肽。这些结果表明,锌还在维护起结构作用的蛋白质结构。 When we introduced mutations at Glu-56 (the proposed active site) and Cys-91 (a proposed catalytic zinc-binding Cys) in BSD, none of the resulting mutants (E56D, E56Q, C91A, C91S, and C91H) showed any detectable activity, as judged with the spectrophotometric assay. Replacements of Cys-91 resulted in gross perturbation of the enzyme structure although the catalytically essential Glu-56 was not necessarily required for proper folding of the enzyme. These results further support our proposal that the catalytic zinc coordinated by the conserved sequence motif is also structural in BSD.