Abstract
Human Pin1, a peptidyl-prolyl isomerase catalyzes the cis/trans isomerization of specifically phosphothreonine/phosphoserine-proline peptide bond, and it is used as molecular switches to regulate cell cycle progression to affect biological activity of protein. Recently it has been found that Alzheimer’s disease may be associated with the dysfunction of human Pin1. In order to avoid the occurrence of the disease or design the drug for treatment, we have to understand the isomerization mechanism of hPin1. We used E.coli expression system to prepare hPin1, and maintained its bioactivity by a moderate purification method. Thermal unfolding measurements indicate hPin1 has a high conformational stability between pH 7 and pH 8, and lowering the pH value will destabilize the protein. We replaced the proline residue in the substrate Ac-ApTPY-pNA with (2S,4S)-hydroproline (hyp), (2S,4R)-hydroproline (Hyp), (2S,4S)-fluoroproline (flp), (2S,4R)-fluoroproline (Flp), (2S,4S)-methoxy-proline (mop), (2S,4R)- methoxyproline (Mop), (2S)-4,4-difluroproline (Dfp), (2S)-4-ketoproline (Kep) to change the ring pucker preference and study the consequence on catalytic efficiency of hPin1. As determined by a protease-coupled assay, replacement of proline with Kep induces a deceased cis content. The cis content of the Hyp, hyp, Dfp peptide was increase upon replacing proline into these derivatives. hPin1 exhibits a better catalytic efficiency toward the flp, Flp, Mop, Dfp peptide than the Pro peptide, and has a lowest catalytic efficiency to the Kep-containing peptide. The results show that a pre-organized ring pucker may not be the key factor contributing to the cis/trans isomer ratio of the peptide and the catalytic efficiency of hPin1. Although the details of the hPin1 catalytic mechanism of cis/trans isomerization is not yet clear, we find that the impacts of the 4-substituded proline derivatives on the catalytic efficiency of hPin1 may be related to steric and hydrophobic effects. Our results may be useful for understanding the catalytic mechanism and the substrate specificity of hPin1.