Detection of weak metal ion binding to cutinase-like enzyme using native mass spectrometry and electron spin...

Weak metal ion binding to enzymes is often necessary for enzyme function; however, its detection remains difficult. The catalytic efficiency of Cut190, a cutinase-like enzyme, is higher with the weak binder Ca²⁺ than with the strong binder Mn²⁺. Upon the introduction of a disulfide bond into Cut190, the enzyme was stabilized and effectively degraded polyethylene terephthalate. Notably, Ca²⁺ binding to the stabilized Cut190 mutant remained weak and was necessary for its function. Here, we demonstrate that native mass spectrometry and electron spin resonance spectroscopy are effective methods for analyzing weak metal ion binding to enzymes.