Myosin-catalyzed ATP hydrolysis elucidated by 31P NMR kinetic studies and 1H PFG-diffusion measurements.

We conducted (31)P NMR kinetic studies and (1)H-diffusion measurements on myosin-catalyzed hydrolysis of adenosine triphosphate (ATP) under varied conditions. The data elucidate well the overall hydrolysis rate and various factors that significantly impact the reaction. We found that the enzymatic hydrolysis of ATP to adenosine diphosphate (ADP) was followed by ADP hydrolysis, and different nucleotides such as ADP and guanosine triphosphate acted as competitors of ATP. Increasing ATP or Mg(2+) concentration resulted in decreased hydrolysis rate, and such effect can be related to the decrease of ATP diffusion constants. Below 50 degrees C, the hydrolysis was accelerated by increasing temperature following the Arrhenius' law, but the hydrolysis rate was significantly lowered at higher temperature (approximately 60 degrees C), due to the thermal-denaturation of myosin. The optimal pH range was around pH 6-8. These results are important for characterization of myosin-catalyzed ATP hydrolysis, and the method is also applicable to other enzymatic nucleotide reactions.