Calorimeter machine in a lab setting

Unlock Enzyme Secrets: How Isothermal Titration Calorimetry Reveals Biological Catalysis

Soraya Malik in Science & Nature August 2025 • 4 min read.

"Delve into the world of enzymatic reactions with isothermal titration calorimetry (ITC), a powerful technique illuminating the dynamics of biological processes."

Life hinges on biochemical reactions, and understanding them unlocks the secrets of biological processes. Calorimetry, a label-free technique, offers a way to quantitatively characterize chemical reactions in solution. It measures heat released or absorbed over time, making it a universal detection system to quantify reacting molecules and reaction rates. This is especially useful for studying enzyme catalysis.

Isothermal Titration Calorimetry (ITC) has become a preferred method for studying the thermodynamics of biomolecular interactions involving proteins, ligands, metal ions, and DNA. ITC's ability to provide kinetic information also makes it a powerful tool to measure enzyme catalysis, an area still being explored for its full potential.

The Michaelis-Menten equation is key to quantitatively describing enzymatic reactions. It shows the relationship between reaction rate and substrate concentration, based on the Michaelis constant (KM) and catalytic rate constant (kcat). The kcat/KM ratio reflects an enzyme's catalytic efficiency. Determining KM and kcat provides a complete description of the catalysis, essential for understanding and manipulating biological processes.

ITC: A Deep Dive into How It Works

Isothermal titration calorimetry measures the heat released or absorbed during a chemical reaction, making it a versatile tool for characterizing various chemical processes. Unlike other methods, ITC doesn't require modifying or labeling the system under analysis and can be performed in solution, using minimal materials. This makes ITC invaluable for studying enzyme kinetics in various fields, including drug discovery.

An isothermal titration calorimeter includes an adiabatic shield with two identical cells. The sample cell holds the enzyme solution, while the reference cell typically contains water or the solvent used in the analysis. A rotating syringe, equipped with a long needle and a stir paddle, introduces a substrate solution into the sample cell. A thermoelectric device measures the temperature difference between the sample and reference cells, maintaining it at zero by adding or subtracting heat.

Substrate Injection: The substrate is injected into the enzyme solution at a constant temperature. When the enzymatic reaction occurs, the amount of heat released or absorbed is directly proportional to the number of substrate molecules converted into product molecules.
Heat Flow Measurement: The rate of heat flow is directly related to the reaction rate. The measured data reflects the thermal power supplied to the sample cell, indicating the heat flow occurring over time.
Data Interpretation: By monitoring the heat changes, researchers can derive crucial kinetic and thermodynamic parameters, providing insights into the enzyme's activity and efficiency.

To obtain a Michaelis-Menten plot, it's essential to measure the total molar enthalpy (ΔH) and the heat flow (dQ/dt) at different substrate concentrations. This is typically achieved through two different experiments. In the first experiment, the substrate is injected into the enzyme solution, and the heat for complete substrate conversion is measured. In the second experiment, multiple injections of the substrate are performed, and the rate of heat production is measured at different substrate concentrations.

ITC: A Reliable Tool for Enzymatic Analysis

ITC provides a reliable and rapid method for characterizing enzymatic reactions by monitoring the heat of reaction without needing system modification or labeling. Unlike traditional methods relying on time-course experiments and spectroscopic measurements, ITC directly measures the heat released or absorbed during the reaction. This eliminates the need for chromophores or coupled assays, reducing potential inaccuracies and simplifying the analysis of enzyme kinetics and inhibition.

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Everything You Need To Know

What is Isothermal Titration Calorimetry (ITC), and how does it work in the context of enzyme reactions?

Isothermal Titration Calorimetry (ITC) is a label-free technique used to study enzymatic reactions by measuring the heat released or absorbed during a reaction. The process involves an adiabatic shield with two identical cells: a sample cell containing the enzyme solution and a reference cell. A syringe injects the substrate into the sample cell. A thermoelectric device measures the temperature difference between the cells and maintains a constant temperature by adding or subtracting heat. The heat flow is directly proportional to the reaction rate, allowing researchers to derive kinetic and thermodynamic parameters, such as the total molar enthalpy (ΔH) and heat flow (dQ/dt), providing insights into enzyme activity and efficiency. The substrate is injected into the enzyme solution at a constant temperature, and when the enzymatic reaction occurs, the amount of heat released or absorbed is directly proportional to the number of substrate molecules converted into product molecules. This makes ITC a powerful tool for characterizing enzymatic reactions and understanding the dynamics of biological processes without the need for modification or labeling.

How does the Michaelis-Menten equation relate to the analysis of enzyme kinetics using ITC?

The Michaelis-Menten equation is crucial in describing enzymatic reactions quantitatively. It shows the relationship between reaction rate and substrate concentration and is based on the Michaelis constant (KM) and the catalytic rate constant (kcat). By using ITC, researchers can measure the heat changes at different substrate concentrations. This data is then used to obtain a Michaelis-Menten plot, which is vital for determining KM and kcat values. These values provide a complete description of the enzyme catalysis, essential for understanding and manipulating biological processes. The kcat/KM ratio reflects the enzyme's catalytic efficiency, further enriching the data from ITC experiments and leading to a deeper understanding of enzyme behavior and function.

What are the advantages of using ITC compared to other methods for studying enzyme kinetics?

ITC offers several advantages over traditional methods. Unlike other techniques, ITC doesn't require modification or labeling of the system under analysis and can be performed in solution, minimizing material use. ITC directly measures the heat released or absorbed during the reaction, eliminating the need for chromophores or coupled assays. This reduces potential inaccuracies and simplifies the analysis of enzyme kinetics and inhibition. Traditional methods often rely on time-course experiments and spectroscopic measurements, which can be less precise and more complex. ITC provides a reliable and rapid method for characterizing enzymatic reactions by directly measuring the heat of the reaction.

Can you explain the specific steps involved in an ITC experiment for enzyme analysis?

An ITC experiment involves the following steps: First, the enzyme solution is placed in the sample cell, and the substrate solution is loaded into a syringe. The reference cell typically contains water or a solvent. Next, the substrate is injected into the enzyme solution at a constant temperature using a rotating syringe. The reaction occurs, and the amount of heat released or absorbed is measured, which is directly proportional to the number of substrate molecules converted. The thermoelectric device measures the temperature difference between the sample and reference cells, adding or subtracting heat to maintain zero difference. The rate of heat flow is directly related to the reaction rate. Finally, researchers monitor heat changes to derive kinetic and thermodynamic parameters, such as total molar enthalpy (ΔH) and heat flow (dQ/dt), ultimately providing insights into the enzyme's activity and efficiency. Multiple injections are often performed to measure heat production at different substrate concentrations.

How does ITC contribute to drug discovery and the understanding of biological processes?

ITC is instrumental in drug discovery and understanding biological processes by providing insights into enzyme kinetics, thermodynamics, and enzyme efficiency. ITC helps researchers characterize the interactions between enzymes and potential drug candidates. By quantifying the heat changes during these interactions, ITC reveals crucial information about binding affinity, binding stoichiometry, and reaction kinetics. These parameters help to identify promising drug candidates and optimize their efficacy. The ability to provide kinetic information makes ITC a powerful tool for measuring enzyme catalysis, providing a complete description of catalysis and helping to manipulate biological processes. ITC's ability to analyze reactions in solution, without labeling, makes it a versatile and valuable technique in various fields, including drug discovery, ultimately advancing our understanding of biological systems and accelerating the development of new treatments.