Beyond IC₅₀: Reframing Microcystin Potency against Protein Phosphatase 2A by Defining Two-Step Irreversible...

Microcystins (MCs) are among the most potent natural inhibitors of serine/threonine protein phosphatases, yet their toxicity is typically assessed using IC₅₀ values that obscure the underlying kinetics and mechanism of enzyme inactivation. Here, we present a comprehensive kinetic analysis of protein phosphatase 2A (PP2A) inhibition by a panel of structurally diverse MC congeners, including MC-LR, MC-LA, MC-LW, MC-RR, [d-Asp³] MC-LR, and the noncovalent control [d-Asp³]-[Dhb⁷] MC-RR. Using an established PP2A inhibition assay method and modeling software to determine K _i, k _inact, and k _inact/K _i, we resolved reversible binding from covalent inactivation to characterize the two-step inhibition mechanism. All covalently competent MCs bind PP2A with picomolar affinity (K _i ≈ 3-11 pM) and undergo slow but tightly constrained covalent modification of Cys269 (k _inact ≈ 3-5 × 10^-4 s^-1), yielding near-diffusion-limited inactivation efficiencies (k _inact/K _i ≈ 10⁷-10⁸ M^-1 s^-1). Structural variation among congeners primarily modulates binding equilibrium and the fraction of complexes that achieve a productive geometry for covalent bond formation, while the irreversible inactivation step remains relatively similar. Notably, [d-Asp³]-[Dhb⁷] MC-RR binds PP2A with picomolar affinity despite lacking a highly reactive electrophile, demonstrating that high-affinity engagement is mechanistically separable from covalent inactivation. These results reveal that MC potency is driven by exceptionally tight and persistent PP2A binding rather than fast reactivity, providing a kinetic framework that links molecular structure to sustained phosphatase inactivation and real-world toxicity during harmful algal bloom exposure.