Characterization and activity-folding relationship of serine protease from Antarctic krill (Euphausia superba)

Abstract

Euphausia superba (Antarctic krill) serine protease (ESP) was investigated to gain insights into the activity-structural relationship, folding behavior, and regulation of the catalytic function. We purified ESP from the krill muscle and characterized biochemical distinctions via enzyme kinetics. Studies of inhibition kinetics and unfolding in the presence of a serine residue modifier, such as phenylmethanesulfonyl fluoride, were conducted. Structural characterizations were measured by spectrofluorimetry, including 1-anilinonaphthalene-8-sulfonate dye labeling for hydrophobic residues. The computational simulations such as docking and molecular dynamics were finally conducted to detect key residues and folding behaviors in a nano-second range. The kinetic parameters of ESP were measured as K_m^BANH = 0.97 ± 0.15 mM and k_cat/K_m^BANH = 4.59 s⁻¹/mM. The time-interval kinetics measurements indicated that ESP inactivation was transformed from a monophase to a biphase process to form a thermodynamically stable state. Spectrofluorimetry measurements showed that serine is directly connected to the regional folding of ESP. Several osmolytes such as proline and glycine only partially protected the inactive form of ESP by serine modification. Computational molecular dynamics and docking simulations showed that three serine residues (Ser183, Ser188, and Ser207) and Cys184, Val206, and Gly209 are key residues of catalytic functions. Our study revealed the functional roles of serine residues as key residues of catalytic function at the active site and of the structural conformation as key folding factors, where ESP displays a flexible property of active site pocket compared to the overall structure.

Communicated by Ramaswamy H. Sarma

Keywords:

• Serine protease
• inhibition
• serine modification
• folding
• Euphausia superba

Acknowledgments

This work was supported by the Key Scientific and Technological Grant of Zhejiang for Breeding New Agricultural Varieties [2021C02069-8-3], the Zhejiang Province Welfare Technology Applied Research Project [LGN21C190013, LGN22C190022], and Key Technology Research and Development Projects in Ningbo [2021Z009]. Jinhyuk Lee was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education [NRF-2020R1I1A2071859] and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program [KGM5362111].

Disclosure statement

The authors declare that they have no conflicts of interest.