Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

Summary

Isothermal titration calorimetry measures heat flow released or absorbed in chemical reactions. This method can be used to quantify enzyme-catalysis. In this paper, the protocol for instrumental setup, experiment running, and data analysis is generally described, and applied to the characterization of enzymatic urea hydrolysis by jack bean urease.

Abstract

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as an intrinsic probe to characterize virtually every chemical process. Nowadays, this technique is extensively applied to determine thermodynamic parameters of biomolecular binding equilibria. In addition, ITC has been demonstrated to be able of directly measuring kinetics and thermodynamic parameters (k_cat, K_M, ΔH) of enzymatic reactions, even though this application is still underexploited. As heat changes spontaneously occur during enzymatic catalysis, ITC does not require any modification or labeling of the system under analysis and can be performed in solution. Moreover, the method needs little amount of material. These properties make ITC an invaluable, powerful and unique tool to study enzyme kinetics in several applications, such as, for example, drug discovery.

In this work an experimental ITC-based method to quantify kinetics and thermodynamics of enzymatic reactions is thoroughly described. This method is applied to determine k_cat and K_M of the enzymatic hydrolysis of urea by Canavalia ensiformis (jack bean) urease. Calculation of intrinsic molar enthalpy (ΔH_int) of the reaction is performed. The values thus obtained are consistent with previous data reported in literature, demonstrating the reliability of the methodology.

Introduction

Quantitative determination of biochemical reactions provides insights into the biological processes at the basis of life. Calorimetry offers a label-free methodology to quantitatively characterize virtually every chemical reaction in solution. This technique measures the heat released or absorbed over time, and is therefore a universal detection system and a very convenient methodology to quantify the amount of reacting molecules (i.e. binding thermodynamics), as well as to measure the reaction rate (i.e. kinetics). In particular, isothermal titration calorimetry (ITC) has been adopted as method of choice to characterize the thermodynamics of biomole....

Protocol

1. Preparing Samples

1. Prepare 2 ml of enzyme solution and 0.5 ml of substrate solution for each experimental run. Dilute concentrated stock solutions of enzyme and substrate in buffer solutions having identical composition to minimize the heat of dilution and mixing during the substrate addition.
   1. Choose the buffer conditions that are adequate to prevent pH change during experiment. For example, 20 mM HEPES pH 7 is adequate for measurements at neutral pH.
      NOTE: If proton exchange is involved, protonation enthalpies of the used buffers must be considered, because they affect the measured ΔH of the reaction. Possible specifi....

Results

Urease (EC 3.5.1.5; urea amidohydrolase) is a multisubunit nickel-containing enzyme found in archea, bacteria, unicellular eukaryotes and plants. This protein acts in the last step of organic nitrogen mineralization, catalyzing the hydrolysis of urea to ammonia and carbamate, which spontaneously decomposes to give a second molecule of ammonia and bicarbonate (Equation 6)¹².

Discussion

Significance of ITC to study enzymatic activity with respect to existing methods

In addition to its classical applications to study binding equilibria, isothermal titration calorimetry provides a reliable and fast method to characterize enzymatic reactions in solution using the heat of reaction as a probe, without requiring system modification or labeling. The kinetic parameters k_cat and K_M are usually obtained through a set of time cour.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HEPES	Sigma	H3375	dissolving in water and adjusting pH with NaOH
TRIZMA-Base	Sigma	T1503	dissolving in water and adjusting pH with HCl
Sodium dihydrogen phosphate	Riedel-de-Haen	4270	dissolving in water
Sodium phosphate dibasic	Riedel-de-Haen	30427	dissolving in water
Urea	Sigma	U4128	dissolving in water at 40 °C
Canavalia ensiformis urease (type C-3)	Sigma	U0251	dissolving in 20 mM HEPES pH 7 and stored at -80 °C
VP-ITC on Origin 7.0	MicroCal (GE Healthcare)	SYS13901	instrument
VPViewer2000 1.30.00 on Origin 7.0	MicroCal (GE Healthcare)		data acquisition software supplied with the instrument