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

We conducted ³¹P NMR kinetic studies and ¹H-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 Mg²⁺ concentration resulted in decreased hydrolysis rate, and such effect can be related to the decrease of ATP diffusion constants. Below 50 °C, the hydrolysis was accelerated by increasing temperature following the Arrhenius’ law, but the hydrolysis rate was significantly lowered at higher temperature (~60 °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.     

Elucidation of spermidine interaction with nucleotide ATP by multiple NMR techniques

Interaction of polyamines with nucleotides plays a key role in many biological processes. Here we use multiple NMR techniques to characterize interaction of spermidine with adenosine 5′‐triphosphate (ATP). Two‐dimensional ¹H‐¹⁵N spectra obtained from gs‐HMBC experiments at varied pH show significant shift of N‐1 peak around pH 2.0–7.0 range, suggesting that spermidine binds to N‐1 site of ATP base. The binding facilitates N‐1 deprotonation, shifting its pK_a from 4.3 to 3.4. By correlating ¹⁵N and ³¹P chemical shift data, it is clear that spermidine is capable of concurrently binding to ATP base and phosphate sites around pH 4.0–7.0. The self‐diffusion constants derived from ¹H PFG‐diffusion measurements provide evidence that binding of spermidine to ATP is in 1:1 ratio, and pH variations do not induce significant nucleotide self‐association in our samples. ³¹P spectral analysis suggests that at neutral pH, Mg²⁺ ion competes with spermidine and shows stronger binding to ATP phosphates. From ³¹P kinetic measurements of myosin‐catalyzed ATP hydrolysis, it is found that binding of spermidine affects the stability and reactivity of ATP. These NMR results are important for advancing the studies on nucleotide–polyamine interaction and its impact on nucleotide structures and activities under varied conditions. Copyright © 2009 John Wiley & Sons, Ltd.