Kinetic and molecular insights into ion-specific regulation of nattokinase by divalent metal ions.

Abstract

Nattokinase (NK), a fibrinolytic serine protease, has attracted attention for biomedical and functional food applications. Divalent metal ions are important determinants of enzyme catalysis and structural stability; however, how metal ions regulate NK at the kinetic and molecular levels remains poorly understood. Elucidating this regulatory mechanism is important not only for understanding the structure-function relationship of NK, but also for the optimization of its production, processing, formulation, and functional performance. In this study, a fibrin knob 'A'-derived fluorescence resonance energy transfer (FRET) substrate was used to quantify metal-ion regulation of NK through steady-state kinetic analysis. Ca2+, Mg2+, Mn2+, Co2+, and Zn2+ were evaluated. Ca2+, Mn2+, Co2+, and Zn2+ enhanced NK catalytic efficiency, with Co2+, Ca2+, Zn2+, and Mn2+ increasing kcat/Kmby approximately 3.29-, 2.83-, 2.78-, and 1.63-fold, respectively, whereas Mg2+ showed minimal effects. These enhancements arose predominantly from reductions in apparent Km rather than changes in kcat, demonstrating that divalent metal ions regulate NK primarily by modulating substrate utilization rather than intrinsic catalytic turnover. Fluorescence quenching and thermodynamic analysis demonstrated that all tested metal ions interacted with NK through spontaneous, predominantly non-covalent interactions, although binding affinity alone did not determine functional effects. Among the ions, Ca2+ uniquely improved thermal stability, and molecular dynamics simulations combined with binding free-energy calculations were consistent with a more stable NK-substrate complex in the presence of Ca2+. Collectively, these findings establish the kinetic basis of ion-specific NK regulation and may inform the optimization of NK activity and stability during production, processing, and functional application.

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