Title:Biochemical Study of Fibrinolytic Protease from Euphausia superba Possessing Multifunctional Serine Protease Activity
Volume: 28
Issue: 6
Author(s): Guo-Ying Qian, Gyutae Lim, Shang-Jun Yin, Jun-Mo Yang, Jinhyuk Lee*Yong-Doo Park*
Affiliation:
- Genome Editing Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Gwahak-ro, Yuseonggu, Daejeon, 34141,Korea
- College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100,China
Keywords:
Fibrinolytic protease, Euphausia superba, kinetics, unfolding, serine residue, osmolytes, molecular dynamics.
Abstract:
Background: Fibrinolytic protease from Euphausia superba (EFP) was isolated.
Objective: Biochemical distinctions, regulation of the catalytic function, and the key residues of
EFP were investigated.
Methods: The serial inhibition kinetic evaluations coupled with measurements of fluorescence
spectra in the presence of 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF) was
conducted. The computational molecular dynamics (MD) simulations were also applied for a comparative
study.
Results: The enzyme behaved as a monomeric protein with a molecular mass of about 28.6 kD
with Km BApNA = 0.629 ± 0.02 mM and kcat/Km BApNA = 7.08 s-1/mM. The real-time interval measurements
revealed that the inactivation was a first-order reaction, with the kinetic processes shifting
from a monophase to a biphase. Measurements of fluorescence spectra showed that serine residue
modification by AEBSF directly caused conspicuous changes of the tertiary structures and exposed
hydrophobic surfaces. Some osmolytes were applied to find protective roles. These results confirmed
that the active region of EFP is more flexible than the overall enzyme molecule and serine,
as the key residue, is associated with the regional unfolding of EFP in addition to its catalytic role.
The MD simulations were supportive to the kinetics data.
Conclusion: Our study indicated that EFP has an essential serine residue for its catalyst function
and associated folding behaviors. Also, the functional role of osmolytes such as proline and glycine
that may play a role in defense mechanisms from environmental adaptation in a krill’s body was
suggested.