Tradler T, Stoller G, Rücknagel K P, Schierhorn A, Rahfeld J U, Fischer G
Forschungsstelle Enzymologie der Proteinfaltung der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Halle/S., Germany.
FEBS Lett. 1997 Apr 28;407(2):184-90. doi: 10.1016/s0014-5793(97)00345-1.
A low degree of amino acid sequence similarity to FK506-binding proteins (FKBPs) has been obtained for the peptidyl prolyl cis/trans 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 side-chain 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 P1 subsites. For the latter enzyme, substitution of Glu178 by Val or Lys, which aligns to Asp37 in human FKBP12, enhanced the PPIase activity, whereas a strongly decreased enzymatic activity was determined for the Asp37Leu and Asp37Val variants of FKBP12. Regardless of the P1 subsite of the substrate used for the assay, mutation of Phe233Tyr 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 Phe99Tyr 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.
已发现大肠杆菌触发因子（TF）的肽基脯氨酰顺/反异构酶（PPIase）结构域与FK506结合蛋白（FKBP）的氨基酸序列相似度较低，人们认为这对于该细菌酶的酶学性质具有重要意义。我们研究了在位置37（FKBP编号）处带负电荷的侧链和位置99处的苯丙氨酸（这两个位点在两种酶中都高度保守）的改变，是否会对一系列具有不同P1亚位点的四肽底物上的FKBP12和TF - PPIase结构域的催化活性产生平行影响。对于后一种酶，将与人FKBP12中Asp37对应的Glu178替换为Val或Lys，增强了PPIase活性，而对于FKBP12的Asp37Leu和Asp37Val变体，酶活性则显著降低。无论用于测定的底物的P1亚位点如何，Phe233Tyr突变产生的TF - PPIase结构域的蛋白质变体的PPIase活性约为野生型的1%。根据底物性质，相应的Phe99Tyr FKBP12变体的k(cat)/K(M)可适度降低，也可增加4.8倍。TF - PPIase结构域的任何一种突变都无法赋予FK506抑制作用，而FK506抑制是FKBP家族PPIases的主要特征。
