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

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.