Background

The study of enzyme kinetics arguably gave rise to the field of biochemistry and remains an important and diverse area of research today. While many important factors are considered when characterizing enzyme kinetics, they ultimately all rely on a robust and accurate means of determining the rate of enzyme-catalyzed product formation under a series of carefully controlled conditions. One important and highly diverse class of enzymes includes those that couple the hydrolysis of ATP to downstream processes such as driving ion pumps, providing mechanical work, unfolding/folding proteins, and supporting otherwise thermodynamically unfavorable chemical reactions (Stock et al., 2000; Sauer et al., 2011; Miller et al., 2016). Despite what processes these enzymes are ultimately responsible for, they are all collectively referred to as ATPases for the critical role ATP hydrolysis plays in their overall function.

The biological importance and diversity of ATPases have driven the development of several methods to quantify ATPase activity. Examples include a wide range of enzyme-coupled assays that rely on the generation of either ADP or inorganic phosphate to support a subsequent enzyme-catalyzed reaction or series of reactions to ultimately generate a product that provides a detectable change in signal (often photon absorbance) (Sehgal et al., 2016; Hoskins et al., 2018). This provides a means for real-time monitoring of ATP hydrolysis though conditions for each enzyme-catalyzed reaction in the scheme must be carefully considered and can limit the utility of this method. Some colorimetric and fluorescence assays have more recently been developed to monitor ATP depletion or product generation without the need for coupling to additional enzyme-catalyzed reactions, but they remain reliant on interaction/reaction with reporter proteins/molecules and can be highly sensitive to reaction buffer composition and contamination (Soyenkoff, 1947; Baykov et al., 1988; Li et al., 2005). Perhaps one of the most robust means for monitoring ATP hydrolysis relies on a radioactive ATP substrate such as γ-³²P ATP or α-³²P ATP. The radioactive ³²P ATP substrate is hydrolyzed by the enzyme, producing either ADP and ³²P (inorganic phosphate) or α-³²P ADP and inorganic phosphate from γ-³²P ATP and α-³²P ATP, respectively (King, 1995). The radioactive products and remaining substrate can then be separated by thin layer chromatography, detected by exposure to either film or a storage phosphor screen and the concentration of product formed quantified as a function of time the reaction was allowed to proceed during the assay. Though this method does not readily support real-time monitoring of product formation, it carries many inherent advantages including high signal-to-noise, capability with essentially any buffer conditions required to support enzyme stability, and no downstream coupling reactions required for product detection/quantitation.

Here, we provide a detailed α-³²P ATP hydrolysis protocol for kinetic characterization of the Shigella type three secretion system ATPase, Spa47 that has provided insight into working with this specific protein (Burgess et al., 2016a and 2016b; Case and Dickenson, 2018). This protocol is intended, however, to serve as a guide for the characterization of any enzymes that hydrolyze ATP as the basis of the protocol is unchanged by the enzyme being studied.