Comparative mutational analysis of peptidyl prolyl cis/trans isomerases: active sites of Escherichia coli trigger factor and human FKBP12

FEBS Letters

Rücknagel, Karl P¹  Rahfeld, Jens-U¹  Stoller, Gerlind¹  Schierhorn, Angelika¹  Tradler, Thomas¹  Fischer, Gunter¹ 

[1] Forschungsstelle ‘Enzymologie der Proteinfaltung’ der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Kurt-Mothes-Str. 3, D-06120 Halle/S., Germany

关键词: Peptidyl prolyl cis/trans isomerase;    Trigger factor (E. coli);    FKBP;    Site-directed mutagenesis;    TF;    trigger factor;    TF-PPIase domain;    trigger factor segment Arg145–Glu251;    PPIase;    peptidyl prolyl cis/trans isomerase;    FKBP;    FK506-binding protein;    FKBP12;    human cytosolic 12 kDa FK506-binding protein;    PMSF;    phenylmethylsulfonyl fluoride;    TLCK;    1-chloro-3-tosylamido-7-amino-l-2-heptanone;    MALDI-MS;    matrix assisted laser desorption ionization-mass spectrometry;    ESI;    electrospray ionization;    TOF;    time-of-flight;   

DOI  :  10.1016/S0014-5793(97)00345-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown