Kinetic characterization of Ca2+-ATPase (SERCA1) inhibition by tri-n-butyltin(IV) chloride. A docking conformation proposal

Abstract

Organotin compounds, such as tri-n-butyltin(IV) chloride (TBT), are widespread toxicants which disrupt different functions in living organisms. TBT interacts with lipid membranes and membrane proteins. The inhibition of the calcium ATPase from sarcoplasmic reticulum membranes by TBT was studied. It was found that the ATPase inhibition could not be reverted in a large time scale; moreover, an excess of TBT over enzyme did not fully inhibit the ATPase activity; therefore, it was concluded that TBT irreversibly inhibits the enzyme, and this inhibition is accompanied by a decrease in the effective TBT concentration. The residual ATP hydrolysis activity was measured at different TBT concentrations with time, and the protective effect of different calcium concentrations on the TBT inhibition was also determined. The simplest kinetic mechanism to successfully explain all the observations and the kinetic behavior was found to be a single irreversible step of the inhibitor binding to the enzyme accompanied with a first-order inhibitor inactivation. A fluorescence study of fluorescein-5-isothiocyanate-labeled enzyme revealed that TBT binding to the enzyme entails a conformational change related to the high- to low-affinity calcium-binding state transition (E1 to E2 transition), resembling the conformational change induced by vanadate linked to the formation of E2 V complex from E1 state. A docking study allowed us to propose a binding pocket for TBT in the membrane region of E1 close to the high-affinity calcium-binding sites, as well as to define the interactions with amino acid residues interfering with calcium sites occupancy.