Background

Kinases and ATPases can be measured by coupling the production of ADP to a spectrophotometrically detectable signal. Commercial vendors provide kits that detect the amount of ADP produced or ATP depleted by coupling them to the production of a fluorescent or bioluminescent signal (examples include ATP-Glo; Promega, ADP-sensor; Biovision). These kits are sensitive but are hard to apply to kinetic questions because they are end-point assays, measuring ADP amounts at a single time point. Furthermore, the black box nature of such kits make it difficult to determine if reaction mixtures are in the steady-state conditions required for Michaelis-Menten kinetics.

Continuous assays measure enzyme activity over time allowing the determination of enzyme rate in a single run. The classic continuous coupled-enzyme assay is the pyruvate kinase and lactate dehydrogenase system (Kornberg and Pricer, 1951). During the reaction, pyruvate kinase uses the ADP produced by the target enzyme to convert phosphoenolpyruvate to pyruvate and in the process regenerate ATP. Pyruvate is then used by lactate dehydrogenase to oxidize NADH to NAD⁺ which is measured by the decrease of absorbance at 340 nm (Figure 1A). A drawback of this method is that in regenerating the ATP continuously, the ATP concentration at any given point is unknown which means that Michaelis-Menten kinetics for ATP cannot be calculated.

An optimal coupled reaction proceeds in a linear pathway so that the ADP produced is converted stoichiometrically into the component producing the signal. ADP-specific hexokinase (ADP-HK) from Thermococcus litoralis uses ADP to phosphorylate glucose into glucose-6-phosphate, which can be used by glucose-6-phosphate dehydrogenase (G6PDH) to reduce NAD⁺ to NADH (Ito et al., 2001; Sakuraba and Ohshima, 2002) (Figure 1B). ADP-HK is an ADP-specific enzyme from thermophilic archaea that is adapted to consume ADP instead of ATP, probably because of the higher thermostability of ADP over ATP. ADP-HK has previously been used in conjunction with G6PDH and diaphorase I to measure the abundance of dNDPs from cellular extracts with a colorimetric signal but has yet to be demonstrated in a continuous assay to measure kinetics (Kumagai et al., 2014). Here, we have used the specificity of ADP-specific hexokinase to develop a method for assaying ADP production by kinases and ATPases.

Figure 1. Schematic of two coupled enzyme assays to measure ATPase/kinase activity. A. The traditional method for measuring ATPase/kinase activity uses pyruvate kinase to couple ADP production to the conversation of phosphoenolpyruvate (PEP) to pyruvate and then lactate dehydrogenase (LDH) to convert pyruvate to lactate–which simultaneously oxidizes NADH to NAD⁺ resulting in a decrease of absorbance at 340 nm. B. Our alternative method uses ADP-HK to couple ADP production to the phosphorylation of glucose which is then used by glucose-6-phosphate dehydrogenase (G6PDH) to convert glucose-6-phosphate to gluconate-6-phosphate–which simultaneously reduces NAD⁺ to NADH resulting in an increase of absorbance at 340 nm.