Metal-cation regulation of enzyme dynamics is a key factor influencing the activity of S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa

Abstract

S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa (PaSAHase) coordinates one K⁺ ion and one Zn²⁺ ion in the substrate binding area. The cations affect the enzymatic activity and substrate binding but the molecular mechanisms of their action are unknown. Enzymatic and isothermal titration calorimetry studies demonstrated that the K⁺ ions stimulate the highest activity and strongest ligand binding in comparison to other alkali cations, while the Zn²⁺ ions inhibit the enzyme activity. PaSAHase was crystallized in the presence of adenine nucleosides and K⁺ or Rb⁺ ions. The crystal structures show that the alkali ion is coordinated in close proximity of the purine ring and a ²³Na NMR study showed that the monovalent cation coordination site is formed upon ligand binding. The cation, bound in the area of a molecular hinge, orders and accurately positions the amide group of Q65 residue to allow its interaction with the ligand. Moreover, binding of potassium is required to enable unique dynamic properties of the enzyme that ensure its maximum catalytic activity. The Zn²⁺ ion is bound in the area of a molecular gate that regulates access to the active site. Zn²⁺ coordination switches the gate to a shut state and arrests the enzyme in its closed, inactive conformation.