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Comparative mutational analysis of peptidyl prolyl cis/trans isomerases: active sites of Escherichia coli trigger factor and human FKBP12
Author(s) -
Tradler Thomas,
Stoller Gerlind,
Rücknagel Karl P,
Schierhorn Angelika,
Rahfeld Jens-U,
Fischer Gunter
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)00345-1
Subject(s) - fkbp , isomerase , peptidylprolyl isomerase , enzyme , biochemistry , active site , chemistry , prolyl isomerase , escherichia coli , tetrapeptide , stereochemistry , biology , peptide , pin1 , gene
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 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.