Inhibition and Activation of Kinases by Reaction Products: A Reporter-Free Assay

Abstract

Kinases are widely distributed in nature and are implicated in many human diseases. Thus, an understanding of their activity and regulation is of fundamental importance. Several kinases are known to be inhibited by ADP. However, thorough investigation of this phenomenon is hampered by the lack of a simple and effective assay for studying this inhibition. We now present a quick, general approach for measuring the effects of reaction products on kinase activity. The method, based on isothermal titration calorimetry, is the first universal, reporter-free, continuous assay for probing kinase inhibition or activation by ADP. In applications to an aminoglycoside phosphotransferase [APH(3′)-IIIa] and pantothenate kinases from Escherichia coli (EcPanK) and Pseudomonas aeruginosa (PaPanK), we found ADP to be an efficient inhibitor of all three kinases, with inhibition constant (K i ) values similar to or lower than the Michaelis−Menten constant (K m ) values of ATP. Interestingly, ADP was an activator at low concentrations and an inhibitor at high concentrations for EcPanK. This unusual effect was quantitatively modeled and attributed to cooperative interactions between the two subunits of the dimeric enzyme. Importantly, our results suggest that, at typical bacterial intracellular concentrations of ATP and ADP (approximately 1.5 mM and 180 μM, respectively), all three kinases are partially inhibited by ADP, allowing enzyme activity to rapidly respond to changes in the levels of both metabolites.

Enzyme Kinetics by Isothermal Titration Calorimetry: Allostery, Inhibition, and Dynamics

“…Thus much less product is produced during a multiple injection assay compared to a single injection one. This is advantageous when strong product inhibition is present (Wang et al, 2019), although conversely, if product inhibition is of interest yet relatively weak, single injection assays would be the more sensitive option. Furthermore, since the data are drawn from post-injection periods where the enzyme velocities have stabilized to constant values, the timescale of the instrument response to changing heat flow can be ignored, simplifying the analysis (as described below).…”

“…It should be noted that single injection assays are usually performed with substantially more dilute enzyme, leading to peaks that are much broader, on the order of 20-60 min, in contrast to the 100 s shown here. Our group has been developing ways to collect and analyze data for rapid single injection experiments, such as those shown in Figure 3C, offering over 10-fold reductions in measurement time and opening the door to new types of experiment (Di Trani et al, 2018b;Wang et al, 2019). Single-injection ITC data can be fitted directly by numerically integrating Equation 2 to give [S](t) and ν 0 (t), and calculating dQ/dt as a function of time according to Equation 6, or using non-linear least squares optimization to find the values of K m and k cat that best reproduce the experimental values.…”

“…The defining feature of this approach is that a full enzyme kinetic characterization is achieved in a single injection. Thus, with substrate in the syringe, it is straightforward to perform many single injection measurements within the same ITC experiment, simply by programming several injections (as many as 10 or 20) spaced at appropriate intervals (Cai et al, 2001;Ertan et al, 2012;Pedroso et al, 2014;Siddiqui et al, 2014;Maximova and Trylska, 2015;Di Trani et al, 2018b;Mason et al, 2018;Abis et al, 2019;Maximova et al, 2019;Wang et al, 2019). For the sake of clarity, we will refer to these as recurrent single injection experiments, to distinguish them from the very different approach termed multiple injection experiments (see section "Multiple Injection Assays, " above).…”

“…Catalytic activity is repeatedly characterized over a period of time, giving information on the stability of the enzyme (Mason et al, 2018). These experiments also provide a sensitive measure of product inhibition (or activation), since the product accumulates in the sample cell with each injection (Cai et al, 2001;Wang et al, 2019). The recurrent single injection approach can be adapted to rapidly characterize other types of inhibition as well, as described below (Di Trani et al, 2018b).…”

“…(E) Data from (C), deconvoluted using the empirical response model (Equation 10), converted to ν 0 and [S] and presented as a double-reciprocal plot. (F) Single injection assay with substrate (ATP) in the syringe and aminoglycoside-3 -phosphotransferase IIIa (APH) and kanamycin A in the sample cell (Wang et al, 2019). Under these dilute conditions [ATP] << K m , ITC peaks decay exponentially with rate constant k eff = k cat /K m .…”