Oxygen Isotopic Exchange Probes of ATP Hydrolysis by RNA Helicases
It is often possible to obtain a detailed understanding of the forward steps in ATP hydrolysis because they are thermodynamically favored and usually occur rapidly. However, it is difficult to obtain the reverse rates for ATP resynthesis because they are thermodynamically disfavored and little of their product, ATP, accumulates. Isotopic exchange reactions provide access to these reverse reactions because isotopic changes accumulate over time due to multiple reversals of hydrolysis, even in the absence of net resynthesis of significant amounts of ATP. Knowledge of both the forward and reverse rates allows calculation of the free energy changes at each step and how it changes when coupled to an energy-requiring conformational step such as unwinding of an RNA helix. This chapter describes the principal types of oxygen isotopic exchange reactions that are applicable to ATPases, in general, and helicases, in particular, their application and their interpretation.