Transthyretin (TTR) is a transporter for thyroid hormone and retinol binding protein that has recently been reported to have proteolytic activity against certain substrates, including amidated neuropeptide Y (NPY). However, the proteolytic activity of TTR towards NPY is not fully understood. Here, we used fluorescence-based assays to determine the catalytic kinetics of human TTR towards human amidated NPY. The Michaelis constant (KM) and catalytic efficiency (kcat/KM) of TTR proteolysis were 15.88 ± 0.44 μm and 687 081 ± 35 692 m -1·s-1, respectively. In addition, we demonstrated an effect of the C-terminal sequence of TTR. When the C-terminal sequence of TTR was made more hydrophobic, the KM and kcat/KM changed to 12.87 ± 0.22 μm and 983 755 ± 18 704 m -1·s-1, respectively. Our results may be useful for the development of TTR as a therapeutic agent with low risk of the undesirable symptoms that develop from amidated NPY, and for further improvement of the kcat/KM of TTR.
Keywords: C‐terminal sequence; amidated neuropeptide Y; fluorescence‐based assay; kinetics; proteolytic activity; transthyretin.