Effect of Divalent Metal Cation on Actomyosin ATPase Kinetics

Abstract

We have used transient time-resolved FRET (TR2FRET), intrinsic fluorescence and fluorescence of pyrene-labeled actin to characterize kinetics of actomyosin cycle with Mn.ATP and Co.ATP ligands. Manganese and cobalt are paramagnetic cations, suitable for high-resolution structural studies of ATP hydrolysis by pulsed electron paramagnetic resonance (EPR). We have shown recently that manganese cation unambiguously reports on the number of coordinated phosphates in the steady state, when myosin is trapped with ADP.AlF4 or AMPPNP. Then, the intimate details of ATP hydrolysis might be studied by transient pulsed EPR. But how relevant is the replacement of natural Mg.ATP with Mn.ATP or Co.ATP in myosin ATPase? The goal of the current study is to determine the effect of divalent 3d-cation on the actomyosin kinetics. We have used previously characterized D.discoideum myosin head construct. We have studied kinetics of the relay helix during the recovery stroke, using TR2FRET. We have monitored kinetics of acto-myosin interaction, using pyrene-labeled actin. We have complemented our study with the data on myosin intrinsic fluorescence, reflecting myosin conformational changes. In our report we compare the rates of ATP binding to actomyosin, ATP hydrolysis, actin binding to myosin in the presence of ADP, and ATP induced dissociation of actomyosin in the presence of ADP for Mn.ATP, Co.ATP, and Mg.ATP, used as a ligand. Supported by NIH AR59621 (YEN).