The conserved methionines of the 1.3 S biotinyl subunit of transcarboxylase: effect of mutations on conformation and activity.
The conserved methionines of the 1.3 S biotinyl subunit of transcarboxylase: effect of mutations on conformation and activity.
复制标题
转羧酶 1.3 S 生物素亚基的保守蛋氨酸：突变对构象和活性的影响。
DOI：
10.1006/abbi.1993.1362
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发表时间：
1993
影响因子：
3.9
通讯作者：
Kumar,GK
中科院分区：
文献类型：
--
作者：
Shenoy,BC;Samols,D;Kumar,GK
来自谢氏丙酸杆菌的转羧酶是一种含生物素的酶，其催化羧基从甲基丙二酰辅酶A可逆转移到丙酮酸。转羧酶26 S复合物由一个中心的六聚体12 S亚基和6个外部的5 S亚基组成，外部连接有12个1.3 S生物素亚基。每个亚基都已克隆并在大肠杆菌中以活性形式表达。我们已经使用克隆的基因在这些亚基的结构-功能相互作用的诱变研究。我们研究的一个特定目标是围绕生物素基赖氨酸的进化上保守的四肽Ala-Met-Bct-Met。我们研究了亚基的性质，每个蛋氨酸（1.3 S M88 L和1.3 S M90 L）含有亮氨酸取代，通过测定它们的活性，在两个部分反应中，该亚基参与。部分反应实验表明，亮氨酸取代对12 S部分反应的影响大于对5 S反应的影响，Met 88的催化作用大于Met 90。为了确定1.3 S突变体中的结构改变是否对活性的影响负责，研究了这些突变体的构象，用胰蛋白酶和V8蛋白酶进行的体外水解研究表明，1.3 S M88 L相对于1.3 S WT和1.3 S M90 L的敏感性存在差异。抗生物素蛋白与1.3 S WT或突变体亚基的复合物的荧光性质表明，1.3 S M88 L的生物胞素的微环境与1.3 S WT和1.3 S M9 OL的生物胞素的微环境不同。相比之下，底物结合（5 S的乙酰丙酮酸和12 S的甲基丙二酰辅酶A）不受任何1.3 S突变体的影响。两者合计，这些结果表明，保守的四肽的1.3 S生物素基亚基，特别是Met 88，是必需的，以提供一个必要的构象和适当的结合性能的催化的部分反应和整体反应。
Transcarboxylase fromPropionibacterium shermaniiis a biotin-containing enzyme which catalyzes the reversible transfer of a carboxyl group from methylmalonyl-CoA to pyruvate. Transcarboxylase 26 S complexes consist of a central, hexameric 12 S subunit with 6 outer, 5 S subunits attached by 12 1.3 S biotinyl subunits. Each of the subunits has been cloned and expressed inEscherichia coliin active form. We have used the cloned genes in mutagenic studies of the structure-function interactions of these subunits. One particular target of our studies has been the evolutionarily conserved tetrapeptide Ala-Met-Bct-Met which surrounds the biotinyl lysine. We have investigated the properties of subunits containing leucine substitutions at each methionine (1.3 S M88L and 1.3 S M90L) by assaying their activity in the two partial reactions in which this subunit participates. Partial reaction assays demonstrate that leucine substitution at either position has a greater effect on the 12 S partial reaction than on the 5 S reaction and Met 88 is more significant catalytically than Met 90. To determine whether structural alterations in the 1.3 S mutants were responsible for the effects on activity, the conformations of these mutants were investigated.In vitrohydrolysis studies with trypsin and V8 protease demonstrated differences in the susceptibility of 1.3 S M88L relative to 1.3 S WT and 1.3 S M90L. Complexes of avidin with 1.3 S WT or mutant subunits, as monitored by fluorescence properties, indicated that the microenvironment of the biocytin of 1.3 S M88L was different from those of 1.3 S WT and 1.3 S M9OL. By contrast, substrate binding (oxalacetate for 5 S and methylmalonyl-CoA for 12 S) was unaffected by any of the 1.3 S mutants. Taken together, these results indicate that the conserved tetrapeptide of the 1.3 S biotinyl subunit, particularly Met 88, is required to provide an essential conformation and proper binding properties for catalysis of the partial reactions and the overall reaction.