Key Points
- Investigate the reaction pathway of acto-heavy meromyosin ATPase to distinguish between single-route and two-route kinetic mechanisms.
- Monitored the binding time-course of the reactive heavy meromyosin (HMM)-phosphate-ADP complex to F-actin via light-scattering intensity in 2 mM MgCl2, 50 mM KCl, and 10 mM Tris-HCl (pH 7.8, ~20°C).
- Measured steady-state ATPase rates across varying ATP and F-actin concentrations using unmodified HMM and HMM treated with CMB and β-mercaptoethanol.
- Determined the extent of acto-HMM dissociation (α) in the presence of ATP using Millipore filtration and light-scattering methods.
- The apparent rate constant k2 increased from 0.16 to 0.41 sec⁻¹ as F-actin concentration increased from 0.1 to 0.75 mg/ml, following first-order kinetics.
- The acto-HMM-ATPase rate peaked at 5 µM ATP before exhibiting substrate inhibition and plateauing above 50 µM ATP; chemical modification of HMM eliminated this substrate inhibition.
- The maximum value of δv0 was 4.25 sec⁻¹ compared to a basal k1 of 0.095 sec⁻¹, with chemical modification altering only the dissociation extent α without modifying rate constants k1, k2, or k3.
Structured PICO
PPopulationIn vitro biochemical model studying the acto-H-meromyosin-ATPase reaction
IInterventionVarying concentrations of ATP and F-actin, and treatment of HMM with CMB and beta-mercaptoethanol
OOutcomeTime-course of binding of the reactive HMM-phosphate-ADP complex with F-actin and rate of acto-HMM-ATPase in the steady statesurrogate
The study provides direct biochemical evidence supporting a two-route mechanism for the acto-myosin-ATPase reaction, contradicting the one-route mechanism.