A fundamental unanswered question in biology is the mechanism of how myosin converts the energy of the hydrolysis of ATP into movement. Another important event in biology is the phosphoryl transfer catalyzed by kinases. Many disorders result from malfunctions of these enzymes. Therefore, the long term objective of this research proposal is to elucidate the nature of the chemical mechanisms of phosphoryl transfers in enzyme- catalyzed reactions.
The specific aims of this proposal are to determine the isotope effects of the ATPase of myosin and a variety of kinases. In order to determine primary and secondary oxygen-18 isotope effects, remotely labeled beta-gamma-[18O]-ATP and gamma-[18O]3-ATP will be synthesized. The remote labeled beta-gamma-[18O]-ATP will be used to survey for primary 18O isotope effects in the non-enzymatic hydrolysis of ATP, as well as in the reactions catalyzed by the ATPase of myosin and a variety of kinases. The results of these primary 18O isotope effects studies will allow us to determine the extent to which the chemistry of phosphoryl transfer is rate limiting in each of these cases. After the conditions for the primary 18O isotope effects have been optimized, we will use gamma-[18O]3-ATP to determine the secondary 18O isotope effects in the gamma- phosphate. The size of the secondary 18O isotope effects will give us information about the nature of the transition state for each enzymatic and non-enzymatic reaction.