The contribution of enzymes as a biological catalyst in almost all processes occurring in live organisms is significant. Studying the enzyme reactions taking place in a system, individually, is essential to gain insights into any biochemical phenomena, with the objective of learning an enzyme’s recognition process of substrates and its approach as a catalyst in the formation of a product. Understanding the mechanism of binding and processing of enzymes with the natural substrate is required to develop next-generation inhibitors.
After obtaining enzymes and substrates, an assay technique needs to be developed to study the enzyme-catalyzed reaction. The combination of these data and structural study data provides insights into the enzyme mechanism. This knowledge is useful to develop potential inhibitors using a rational approach. However, it is a challenging process to develop suitable assays.
The Solution - High-Sensitivity ITC
High-sensitivity ITC is a proven and robust technique that can address the challenge by allowing quantitative observation of enzymatic reactions to get the associated Michaelis-Menten kinetic constants describing the system. As a versatile instrument, ITC is increasingly used to quantify the thermodynamics of equilibrium association reactions.
ITC provides measurements of the binding enthalpy (ΔHB) changes for reversible interactions directly and without using models. Moreover, the equilibrium association constant (KA) for the enzyme-substrate complex can be roughly calculated using well-designed experiments, where the limitation step is the product formation and the stoichiometry (n) of the reaction. Hence, nearly complete thermodynamic profiles can be obtained for any bimolecular complex formation with the help of ITC.
ITC is a widely used assay technique for the analysis of any enzyme- catalyzed reaction, provided a heat signal is associated with the reaction. It takes less time to complete the experiments (typically ~2 hours) and consumes less amount of material. This article explores the application of ITC in the analysis of catalytic reactions and demonstrates the analysis method of ITC-derived enzymatic reaction data to determine enzyme kinetic parameters.
Calorimetry and Kinetics
Enzyme kinetic parameters can be measured with ITC owing to the fact that the thermal energy released during the reaction is a measurable event. The reaction rate varies in proportion to the thermal power, which has been expressed as a function of time (dt): Power = dQ/dt. MicroCal VP-ITC has a minimum response time of 15s and is extremely fast to study the enzyme reaction rate of many processes, avoiding the need to correct the measurable thermal power for the time constant of the calorimeter.
The ITC’s normal titration mode allows making multiple injections of substrate, enabling to determine multiple rates within a single experiment under steady-state conditions. In addition, modern ITC instruments are extremely sensitive, requiring an enzyme quantity similar to spectrophotometric assays but more than radioactivity assays. The amount of heat generated in the conversion of ‘n’ moles of substrate to product is expressed as follows:
Where, ΔHapp = total molar enthalpy for the reaction that is experimentally determined; P = concentration of product generated; and V = Volume of the reaction solution (cell volume).
The thermal power is expressed as follows:
The reaction rate can be obtained by rearranging the above equation as follows:
The reaction rate equation reveals that it is necessary to determine the total molar enthalpy and the power generated (dQ/dt) using the calorimeter to get a Michaelis-Menten plot. The analysis of enzyme-catalyzed reactions using calorimetry simultaneously provides Michaelis-Menten based kinetic and thermodynamic data.
ITC is simple but robust analytical instrument allowing the manipulation of all of the above experimental conditions to perform a systematic characterization of an enzyme system economically and without labelling. Making up the reactants in suitable and well-defined buffer systems is crucial for all ITC experiments. The molar concentration of both the substrate and enzyme needs to be accurately measured to carry out ITC enzymatic assays using ITC. Determining the apparent molar enthalpy is essential for an ITC assay. It is also advantageous to determine the exact molar concentration of active enzyme. Accurate quantification of substrate concentrations in solutions is also required.
It is necessary to perform two types of experiments in order to carry out an ITC assay. The first experiment is the determination of the total molar enthalpy by holding comparatively large amounts of enzyme in the cell and the substrate in fairly low quantity in the injection syringe to provide sufficient space between injections for conversion of all of the substrate to product. ΔHapp can be obtained in the normal way using the resultant peaks.
The second experiment is the determination of the reaction rate data by holding fairly low concentrations of enzyme in the cell, comparatively high concentrations of substrate in the injection syringe to provide small gaps between injections. The objective is to maintain steady-state conditions and ensure that no more than 5% of the substrate is depleted before the next injection. From these two data sets, the Michaelis-Menten defined enzyme parameters KM, kcat and Vmax can be derived.
This example presents the enzyme rate data for a serine/threonine phosphatase (PP1- γ, see Figure 1). ITC was used to analyze this system. Using the enzyme illustrated in Figure 1 and PNPP as substrate, the aforementioned two types of ITC analysis was performed. The assay results that are useful to determine the rate data for this enzyme- catalyzed reaction are presented in Figure 2.
Figure 1. X-ray crystal structure of the catalytic subunit of PP1 (from Reference 14); the active site His, Asn and Asp residues are shown. (B) Active site of PP1 showing the dinuclear octahedrally coordinated metal ion core with bridging inorganic phosphate (product).
Figure 2. Raw calorimetric data for the measurement of the reaction rate for the hydrolysis of PNPP by PP1-γ. The dashed line is a linear least squares best fit to the preinjection baseline.
A Microsoft™ Excel™ spreadsheet was initially used to analyze the results presented in Figure 2. The non-linear least squares analysis package in Origin™ 5.0 was used to fit the rate data. From these data, ΔHapp was quantified separately. The analysis results for the PPI-γ-PNPP system are presented in Figure 3. Heats of substrate dilution are represented in green in the upper panel.
Figure 3. In the top panel the red peaks are raw calorimetric data for the determination of ΔHapp for the hydrolysis of PNPP by PP1- γ. Integration of these peaks gives the data in the bottom panel.
The main objective of this analysis was to validate ITC for application in kinetic assays for this enzyme system, and compare ITC-derived enzymatic parameters with the values obtained from other assays. For this reason, PNPP was selected as a model substrate, which already has a well-established spectroscopic assay. The calorimetric data were assessed by performing spectroscopic assays in a manner similar to ITC assays.
The spectroscopic assay was employed to derive initial velocities at many different substrate concentrations to build a Michaelis-Menten plot. Figure 4 presents the combined ITC and spectroscopic data. Two sets of data are presented that are normalized for enzyme concentration. First is the rate data from ITC assays (represented as blue line) and second is the analogous data from spectrophotometry assays (represented as red line). The blue line represents a non-linear least squares best fit to the spectroscopic data, fitted using the the following equation:
Figure 4. Rate vs substrate concentration data for the hydrolysis of PNPP by PP1- γ.
The results clearly demonstrate the advantage of using ITC for quantitative enzyme kinetics. For the given system, the ITC assay data correlates well with normal spectroscopic assay data, demonstrating the supremacy of ITC over other methods. Besides providing quality enzymatic rate data, calorimetry measures the thermodynamics of the reaction. This feature of the ITC assay holds potential to provide quality and extensive data about enzyme-catalyzed reactions.
This information has been sourced, reviewed and adapted from materials provided by Malvern Panalytical.
For more information on this source, please visit Malvern Panalytical.