Comparative mutational analysis of peptidyl prolyl cis/trans isomerases: Active sites of Escherichia coli trigger factor and human FKBP12
Comparative mutational analysis of peptidyl prolyl cis/trans isomerases: Active sites of Escherichia coli trigger factor and human FKBP12
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DOI：
10.1016/s0014-5793(97)00345-1
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发表时间：
1997-04-28
期刊：
影响因子：
3.5
通讯作者：
Fischer, G
中科院分区：
文献类型：
--
作者：
Tradler, T;Stoller, G;Fischer, G
大肠杆菌触发因子 (TF) 的肽基脯氨酰顺反异构酶 (PPIase) 结构域与 FK506 结合蛋白 (FKBP) 具有低程度的氨基酸序列相似性，这被认为对细菌酶的酶学特性具有重要意义。我们检查了第 37 位（FKBP 编号）处的带负电侧链和第 99 位的苯丙氨酸是否发生了高度的改变。两种类型的酶都保守，导致对一系列具有不同 P-1 亚位点的四肽底物中的 FKBP12 和 TF-PPIase 结构域的催化活性产生平行影响。对于后一种酶，用 Val 或 Lys 取代 Glu(178)（与人 FKBP12 中的 Asp(37) 对齐），增强了 PPIase 活性，而确定了FKBP12 的 Asp(37)Leu 和 Asp(37)Val 变体，无论用于测定的底物的 P-1 亚位点如何，Phe(233)Tyr 的突变都会产生 TF-PPIase 结构域的蛋白质变体，其活性约为野生型 PPIase 活性的 1%。根据底物性质，可以确定相应的 k(cat)/K-M 适度降低以及 4.8 倍增加。 Phe(99)Tyr FKBP12 变体，TF-PPIase 结构域的两种突变均无法植入 FK506 抑制，而 FK506 抑制是 FKBP PPIase 家族的主要特征。 (C) 1997 年欧洲生化学会联合会。
A low degree of amino acid sequence similarity to FK506-binding proteins (FKBPs) has been obtained for the peptidyl prolyl cisltrans isomerase (PPIase) domain of E. coli trigger factor (TF) that was thought to be significant with regard to the enzymatic properties of the bacterial enzyme, We examined whether the alteration of a negatively charged sidechain at position 37 (FKBP numbering) and a phenylalanine at position 99, both highly conserved through both types of enzymes, leads to parallel effects on the catalytic activity of both FKBP12 and TF-PPIase domain in a series of tetrapeptide substrates with different P-1 subsites, For the latter enzyme, substitution of Glu(178) by Val or Lys, which aligns to Asp(37) in human FKBP12, enhanced the PPIase activity, whereas a strongly decreased enzymatic activity was determined for the Asp(37)Leu and Asp(37)Val variants of FKBP12, Regardless of the P-1 subsite of the substrate used for the assay, mutation of Phe(233)Tyr generated a protein variant of the TF-PPIase domain with about 1% of the wild type PPIase activity, Dependent on the substrate nature, a moderate decrease as well as a 4.8-fold increase in k(cat)/K-M could be determined for the corresponding Phe(99)Tyr FKBP12 variant, Neither of the mutations of the TF-PPIase domain was able to implant FK506 inhibition found as a major characteristic of the FKBP family of PPIases. (C) 1997 Federation of European Biochemical Societies.