The Specialty Fertilizer Products Company (SFP) is acknowledged for providing the funds necessary for this study.

1. Preparing Samples
<ol>
	<li>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.
	<ol>
		<li>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 &#916;H of the reaction. Possible specific effects of the buffer or additives molecules on the system under analysis must be taken into account. If organic solvents (e.g. DMSO) are included in the enzyme solution, add them at exactly the same concentration into the substrate solution.</li>
		<li>For the M1 experiment, start with enzyme concentration in the nM range (e.g. 1 nM) and with a substrate concentration at least three orders of magnitude higher than the enzyme concentration and above the KM.</li>
		<li>For the M2 experiment, start with enzyme concentration in the pM-nM range (e.g. 15 pM). Substrate concentration in the syringe is in the mM range (e.g. 400 mM). 
		NOTE: In the single injection M1 experiment, concentrations should be adequate to achieve the total substrate conversion into the product over the experimental time. Therefore, the concentration of the enzyme depends on the enzyme rate: if enzymes with low kcat are under study, higher enzyme concentrations (up to 10 &#181;M) must be used. On the other hand, enzyme concentrations used in the M2 experiment must assure that the injected substrate is only marginally (less than 5%) consumed and that the enzyme reaction proceeds at the steady state. For this reason, the higher the enzyme efficiency, the lower the required enzyme concentration. At the end of the experiment, substrate concentration in the sample cell should be higher than KM.</li>
	</ol>
	</li>
	<li>Carefully check enzyme and substrate concentrations with an appropriate analytical procedure (e.g. absorbance at 280 nm, colorimetric, BCA assays11. This is required to obtain accurate and reliable calculation of the thermodynamic and kinetic parameters.</li>
	<li>Measure the pH of the solutions and verify that the pH mismatch of both the enzyme and the substrate solutions is minimal under the experimental conditions (&#177;0.05 pH units).</li>
</ol>
2. Performing the Experiment
NOTE: The same procedure must be applied both for the M1 and the M2 experiment, which are performed one after the other.
<ol>
	<li>Verify that the sample cell and the injection syringe are cleaned according to the manufacturer&#8217;s instructions. Fill the loading syringe provided with the instrument with distilled water, gently insert the needle in the sample cell, fill the cell and remove the liquid using the same syringe. Using this method, wash the sample cell twice with distilled water and twice with the buffer.</li>
	<li>Load the sample cell with 2 ml of enzyme solution using the loading syringe, carefully avoiding formation of air bubbles. Slowly inject the solution into the cell until it spills out the top of the cell stem. Produce a spurt of ca. 0.25 ml of solution into the cell. Repeat twice. This step removes air bubbles trapped in the sample cell.</li>
	<li>Place the needle of the loading syringe on the ledge between the cell stem and the cell port and remove any excess solution.</li>
	<li>Start the VP-Viewer program and, from the computer interface, equilibrate the ITC instrument to a temperature 3 &#176;C below the desired experimental temperature. This is required to avoid long equilibration periods before the experiment, due to the passive instrumental cooling device.</li>
	<li>Fill the reference cell with distilled water with the same procedure above. When buffers with high ionic strength or high osmolality are in the sample solution, the same buffer should be used as a reference solution.</li>
	<li>Link a plastic syringe to the fill port of the injection syringe, using a thin silicon tube. Fill the injection syringe placing the needle tip into water and drawing up, until water comes out of the top filling port, indicating that the injection syringe is full. Then move the syringe tip from water and draw up air, to empty the injection syringe. With this procedure, wash the injection syringe with buffer, and draw air through the system. Subsequently, place the injection needle in a narrow tube containing 0.5 ml of substrate solution, carefully draw up and completely fill the injection syringe, leaving small amount of solution at the bottom of the tube.</li>
	<li>From the computer interface, press &#8220;Close fill port&#8221;. Remove the silicon loading tube. Press &#8220;Purge and refill&#8221; button to allow the injection syringe to dislodge any air bubbles and to expel them back into the bulk solution. Repeat twice.</li>
	<li>Move the injection syringe, wipe on the sides to remove any drops and place the needle of the injection syringe into the sample cell.</li>
	<li>On the computer, set the appropriate running parameters. The experimental enzyme and substrate concentrations can be indicated.
	<ol>
		<li>In the M1 experiment, set at least two additions (5-30 &#181;l) of substrate to verify the reproducibility. Set the spacing time between each injection (e.g. 1,000 sec) large enough to ensure that the heat signal returns to the baseline before the next addition.</li>
		<li>In the M2 experiment, set multiple (e.g. 15 x 5-10 &#181;l) injections. Set the interval between the injections (e.g. 180 sec) allowing the system to stabilize the thermal power to the new baseline after each injection. 
		NOTE: The spacing time between injections in the M2 experiment should be short enough to avoid the conversion of a significant amount of substrate, allowing the measurements to be performed under steady state conditions.</li>
		<li>In the M2 experiment, use small volumes (e.g. 2 &#956;l) for the first injection, whose corresponding value is discarded during the subsequent data analysis. Indeed, it often presents artifacts due to the initial substrate diffusion through the syringe tip, and for the presence of air bubbles trapped in the syringe needle.</li>
		<li>Set the reference power, the approximate level in which the baseline will be placed before the reaction starts, at a value of 20. Then define the experimental enzyme and substrate concentrations and chose a name for the experiment.</li>
		<li>Define the experimental temperature, typically at 25 &#176;C. ITC allows working temperatures between 2 &#176;C and 80 &#176;C. The experiment is ready for running. Press &#8220;Start&#8221; button to start the experiment.</li>
	</ol>
	</li>
	<li>Once the experiment has finished, clean the sample cell and the syringe according to the manufacturer&#39;s instructions.</li>
	<li>Repeat the experiment at least one or two more times to check the reproducibility of the data.</li>
</ol>
3. Data Analysis
<ol>
	<li>From the analysis program, click on the &#8220;Read data&#8221; button, and navigate to the folder where the .itc file of the performed M2 experiment is located. Click on the scroll down arrow of the &#8220;Files of type&#8221; and select &#8220;Enzyme Assay (it?)&#8221;. Subsequently, click and open the&#160;.itc file of the M2 experiment.</li>
	<li>Obtain the &#916;H of the reaction from integrating the curve of the M1 experiment according to Equation 5. 
	<img fo:content-width="1.5in" src="/files/ftp_upload/51487/51487eq5.jpg" width="126" /> (5)
	<ol>
		<li>Click on the &#8220;Read data&#8221; button, and select the file .itc of the performed M1 experiment. Using Origin, divide the trace in different parts containing one peak each and save each peak as an .opj file. Open the file corresponding to the first peak, resulting from baseline deviation in the thermogram of the single injection M1 experiment, integrate it and divide the obtained area value, expressed in &#181;cal, by the final substrate concentration in the sample cell, expressed in &#181;M, and by the cell volume expressed in liters, determining, according to Equation 5, the &#916;H of the reaction.</li>
		<li>Repeat the same procedure for the second peak of the M1 experiment and obtain an average value for the two &#916;H measurements.</li>
	</ol>
	</li>
	<li>Determine dQ/dt from the M2 experiment measuring the difference between the original baseline and the new baseline following each injection. Convert the experimental data to reaction rates according to Equation 4, using the enthalpy value obtained in the M1 experiment and fit the data to the Michaelis-Menten equation.
	<ol>
		<li>From the analysis program, click on the &#8220;Read data&#8221; button, and select the file .itc of the performed M2 experiment. Click on the scroll down arrow of the &#8220;Files of type&#8221; and select &#8220;Enzyme Assay (it?)&#8221;.</li>
		<li>The Enzyme Assay dialog box opens, allowing selecting one of the four models. Select &#8220;Method 2 - Substrate only&#8221; from the window.</li>
		<li>In the &#916;H window, indicate the value of &#916;H obtained in step 4.2.</li>
		<li>Click the &#8220;Concentration&#8221; button to check the concentration values to be used in the fitting procedure. Click the &#8220;Average Time (P)&#8221; button. The dialog box opens giving the opportunity to change or accept the default value. This value represents the time before each injection in which the instrument averages the power signal to determine the power level at each substrate concentration. Click OK to confirm the default value.</li>
		<li>Click the &#8220;Zero Y Axis&#8221; button. The cursor turns to a cross hair allowing to double click a point, to place at y=0. Double click just before the first injection point.</li>
		<li>Click the Calculate Rate button. Rate is plotted versus the substrate concentration, derived by dividing the substrate added during the titration for the sample cell volume (taking into account dilution effects). This operation gives a typical Michaelis-Menten plot that can be fitted to obtain KM and kcat.</li>
		<li>If some points should be deleted, select the &#8220;Truncate Data&#8221; button. Move the data markers to exclude the bad data points and double click on one of the markers or press enter.</li>
		<li>Use the &#8220;Fit to model&#8221; function to fit the curve and to obtain the kinetic constants.</li>
	</ol>
	</li>
</ol>

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A., Huber, R., Poulos, T., Wieghardt, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Metalloproteins. , 867-879 (2001).</li><li>Goldberg, R., Kishore, N., Lennen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamic+quantities+for+the+ionization+reactions+of+buffers.">Thermodynamic quantities for the ionization reactions of buffers.</a> J. Phys. Chem. Ref. Data. 31, 231-370 (2002).</li><li>Baumann, M. 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M., Niemann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+competitive+inhibition+of+the+urease-catalyzed+hydrolysis+of+urea+by+phosphate.">The competitive inhibition of the urease-catalyzed hydrolysis of urea by phosphate.</a> J. Biol. Chem. 177, 601-605 (1949).</li><li>Benini, S., Rypniewski, W. R., Wilson, K. S., Ciurli, S., Mangani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-based+rationalization+of+urease+inhibition+by+phosphate:+novel+insights+into+the+enzyme+mechanism.">Structure-based rationalization of urease inhibition by phosphate: novel insights into the enzyme mechanism.</a> J. Biol. Inorg. Chem. 6, 778-790 (2001).</li><li>Segel, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Enzyme kinetics: behavior and analysis of rapid equilibrium and steady-state enzyme systems. , (1993).</li></ol>

The authors have nothing to disclose.

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 kcat and KM are usually obtained through a set of time course experiments, in which the product formation (or substrate consumption) is monitored in continuous or discontinuous assays. Typically, the substrate or product concentration is measured by light absorbance or fluorescence at characteristic wavelengths. However, most substrates and/or products are not spectroscopically active, and therefore a chromophore or fluorophore must be included to follow the reaction. Alternatively, coupled assays, with the product of the catalysis being a substrate for another reaction with measurable product, could be employed. These methods, however, introduce additional variables that reduce the accuracy of the determined values of KM and kcat. In addition, the characterization of enzyme inhibition by small ligands using traditional methods could be complicated by the absorbance or fluorescence of the small ligand itself, which may alter the reliability of the spectroscopic assay. Alternative methods involve stopped flow techniques, in which the amount of product is quantified using mass spectrometry or chromatography. These techniques are very precise and reliable, but are time consuming and expensive for routine analysis. The probe followed in ITC experiments, that is the heat released or absorbed, is an intrinsic property of almost all chemical reactions. Therefore ITC does not require any modification or labeling of the system under analysis. Moreover, this technique is straightforward and fast, requires small amount of material and can be performed in solution22,23.
Critical steps within the protocol and possible modifications
The reaction analysis is generally performed in two different experiments, which provide the total molar enthalpy of the reaction and the heat change over time at different substrate concentrations. In order to obtain good quality data, the experiment must be planned in detail at the beginning of the investigation. The protein and substrate concentrations must be carefully determined, as their correct value is essential for calculating reliable parameters in data analysis. The buffer composition and the pH of both the enzyme and the substrate solutions must be identical, in order to minimize heat of mixing during substrate addition. This is generally ensured by performing a dialysis or a size exclusion chromatography step on the enzyme solution before running the experiment, and using the buffer eluting from the column to dissolve the substrate. A control experiment must be performed with identical solutions of substrate injected into the reaction buffer in the absence of the enzyme, in order to subtract the heat of dilution or to verify that it is negligible.
Data analysis is generally performed with the Origin program for ITC provided by the manufacturer. An alternative program of choice for data analysis can be employed, if needed.
Troubleshooting and limitations of the technique
A good signal-to-noise ratio is obtained only if the reaction proceeds at a rate that allows the system to produce enough heat in the ITC cell to overcome the instrumental detection limit. This is usually achieved for systems with kcat higher than 1 min-1. In this case, the Origin software for ITC automatically calculates the Michaelis-Menten plot from raw data, and using nonlinear least-squares regression analysis, it calculates kcat and KM values. This analysis assumes no significant product inhibition. The latter is generally visible when subsequent injections in the M1 experiment produce peaks with different shapes and, in particular, with the power trace of the second peak taking longer to return to the initial baseline. If product inhibition occurs, another software of choice, implemented with purpose-specific equations, must be used.
Given the intrinsic difference of every enzymatic system, and the different heat produced by different reactions, which cannot be quantified a priori, determining the optimal running conditions (i.e. enzyme and substrate concentrations, number and volume of injections, spacing time, etc.) for a specific system sometimes require few exploratory titrations.
During the analysis, care must be applied to choose the experimental conditions according to the system under analysis. For example, phosphate inhibits urea hydrolysis by urease, and therefore, performing the experiment in phosphate buffer decreases the total rate of the enzymatic reaction24,25.
Applications
Once the kinetics of enzymatic reaction has been determined, the reaction can be repeated in the presence of different types and concentrations of enzyme inhibitors in the sample cell, in order to measure, using the general inhibition Equation 8&#160;26, competitive (Kic) and uncompetitive (Kuic) inhibition constants, thus allowing us to distinguish, among different types of inhibition.
<img fo:content-width="3in" src="/files/ftp_upload/51487/51487eq8.jpg" width="284" /> (8)
In conclusion, this method can be used as a fast and economic way to test a large number of enzyme inhibitors for pharmaceutical and industrial applications, such as in drug screening.

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 biomolecular equilibria, involving protein-ligand, protein-protein, protein-metal ions and protein-DNA interactions1-6. In addition, the ability of ITC to provide kinetic information makes it a very powerful system to measure enzyme catalysis, although the potential of this application is still underestimated7-9.
The Michaelis-Menten equation10 is a quantitative description of enzymatic reactions, as it provides a relationship between the reaction rate and the substrate concentration, depending on two kinetic parameters: the Michaelis constant (KM) and the catalytic rate constant (kcat). The kcat/KM ratio is referred as the catalytic efficiency of an enzyme. In practice, determination of KM and kcat for a specific reaction provides a complete description of the catalysis.
In a typical enzymatic reaction (Figure 1), a substrate (S) interacts with the enzyme (E) forming the enzyme-substrate (ES) complex, which is subsequently activated into the transition state (ES*). The latter is converted into the enzyme-product (EP) complex that eventually dissociates. These steps are described by the following reaction.
<img fo:content-width="2in" src="/files/ftp_upload/51487/51487eq1.jpg" width="197" /> (1)
where k1 is the rate constant for the formation of the ES complex, k-1 is the rate constant for the dissociation of the ES complex, while kcat is the catalytic rate constant or turnover number.
According to the Michaelis-Menten equation10, the rate of the reaction can be calculated as:
<img fo:content-width="1.5in" src="/files/ftp_upload/51487/51487eq2.jpg" width="144" /> (2)
in which KM = (k-1 + kcat)/k1 and kcat = vmax/[E], with vmax being the maximal velocity reached when all enzyme is bound to the substrate.
The isothermal titration calorimeter is the instrument used in this study to characterize the enzymatic hydrolysis of urea. This instrument is made of an adiabatic shield containing two coined-shaped cells (Figure 1). These are connected to the outside with narrow access tubes. The sample cell (ca. 1.4 ml) is loaded with the enzyme solution, while the reference cell is generally filled with water or with the solvent used for the analysis. A rotating syringe with a long needle and a stir paddle attached, usually containing ca. 0.3 ml of substrate solution, is mounted on the sample cell. A thermoelectric device measures the difference of temperature between the sample and the reference cell and, using a &#8220;cell feedback network&#8221;, it maintains this difference at zero by adding or subtracting heat. During the experiment, the substrate is injected into the enzyme solution at a constant chosen temperature. When the enzymatic reaction takes place, the amount of heat released or absorbed is proportional to the number of substrate molecules that are converted into product molecules. In addition, the rate of heat flow is directly related to the rate of the reaction. The measured data, appearing as a deviation of the heat trace from initial baseline (Figure 1), represent the thermal power (&#956;cal/sec) supplied by the instrument to the sample cell, which is proportional to the heat flow occurring in the sample cell over time.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51487/51487fig1highres.jpg" width="500" /> 
Figure 1. Schematic representation of isothermal titration calorimeter to study enzymatic reactions. An enzymatic reaction occurring upon titration of the substrate (in the injection syringe) into the enzyme solution (in the sample cell) results in a change of the thermal power released by the calorimeter, needed to keep the difference of temperature between the sample cell and the reference cell constant. <a href="https://www.jove.com/files/ftp_upload/51487/51487fig1highres.jpg" target="_blank">Click here to view larger image.</a>
Overall, the heat change (Q) is proportional to the molar enthalpy of the reaction (&#916;H) and the number of moles of product generated (n), which in turn is given by the total volume times the concentration:
<img fo:content-width="2in" src="/files/ftp_upload/51487/51487eq3.jpg" width="200" /> (3)
The product formation over time (dP/dt), which corresponds to the reaction rate, can thus be related to the amount of heat generated over the same time (dQ/dt) through the relation:
<img fo:content-width="2in" src="/files/ftp_upload/51487/51487eq4.jpg" width="150" /> (4)
According to this equation, in order to obtain a Michaelis-Menten plot it is necessary to measure i) the total molar enthalpy &#916;H, and ii) the heat flow dQ/dt at different substrate concentrations. Usually, this is performed in two different experiments: in the first experiment (Method 1, M1), the substrate is injected into the enzyme solution and the heat for complete substrate conversion is measured; in the second experiment (Method 2, M2), multiple injections of substrate are performed and the rate of heat production is measured at different substrate concentrations. These two sets of data are sufficient to derive the kinetic parameters KM and kcat.
In the present article, a general protocol to determine the kinetic parameters for enzymatic reactions performed using ITC is described. We applied the method to urea hydrolysis by Canavalia ensiformis urease, as a reference system. The good agreement between the results obtained using this method and the data reported in literature demonstrates the reliability of this approach.

<table border="0" cellpadding="0" cellspacing="0" width="796">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">HEPES</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">H3375</td>
			<td style="width:323px;">dissolving in water and adjusting pH with NaOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">TRIZMA-Base</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">T1503</td>
			<td>dissolving in water and adjusting pH with HCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sodium dihydrogen phosphate</td>
			<td style="width:111px;">Riedel-de-Haen</td>
			<td style="width:119px;">4270</td>
			<td>dissolving in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sodium phosphate dibasic</td>
			<td style="width:111px;">Riedel-de-Haen</td>
			<td style="width:119px;">30427</td>
			<td>dissolving in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Urea</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">U4128</td>
			<td>dissolving in water at 40 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Canavalia ensiformis urease (type C-3)</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:119px;">U0251</td>
			<td>dissolving in 20 mM HEPES pH 7 and stored at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;"></td>
			<td style="width:111px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">VP-ITC on Origin 7.0</td>
			<td style="width:111px;">MicroCal (GE Healthcare)</td>
			<td style="width:119px;">SYS13901</td>
			<td>instrument&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">VPViewer2000 1.30.00 on Origin 7.0</td>
			<td style="width:111px;">MicroCal (GE Healthcare)</td>
			<td style="width:119px;"></td>
			<td>data acquisition software supplied with the instrument</td>
		</tr>
	</tbody>
</table>

hot biological catalysis: isothermal titration calorimetry to characterize enzymatic reactions

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 (kcat, KM, &#916;H) 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 kcat and KM of the enzymatic hydrolysis of urea by Canavalia ensiformis (jack bean) urease. Calculation of intrinsic molar enthalpy (&#916;Hint) of the reaction is performed. The values thus obtained are consistent with previous data reported in literature, demonstrating the reliability of the methodology.

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.
<img fo:content-width="4in" src="/files/ftp_upload/51487/51487eq6.jpg" width="450" /> (6)
This reaction causes an overall increase of the pH of the environment, which is responsible of negative effects for human health and agriculture13. In plants, the primary role of urease is to recycle nutrient nitrogen from arginase-derived urea14. Plant ureases are generally homo-hexamers a6 with each subunit containing an active site with two Ni2+ ions15. Among them, Canavalia ensiformis (jack bean) urease (JBU) has been the first protein to be crystallized in 192616. Since then, several studies have extensively characterized the structural and enzymatic activity of ureases from several sources13,17. Therefore, the ureolytic activity of JBU was chosen as a representative reaction to demonstrate the applicability of ITC in quantitatively determining enzymatic catalysis. A value of &#916;H = -10.5 kcal/mol&#160;was measured in a M1 experiment (Figure 2A) by integrating the thermal power resulting by injecting urea substrate into the urease solution and allowing the reaction to proceed to completion. The thermal power registered in a M2 experiment (Figure 2B) was converted to the reaction rate using Equation 4 (Figure 2C). Fit of the obtained data to the Michaelis-Menten equation provided the kinetic parameters for JBU in 20 mM HEPES pH 7 as kcat = 30,200 sec-1 and KM = 3.45 mM, in agreement with previously reported data18,19.
<img alt="Figure 2" fo:content-width="2.5in" src="/files/ftp_upload/51487/51487fig2highres.jpg" width="500" /> 
Figure 2. Urea hydrolysis by urease determined by ITC. JBU (type C3) was purchased as lyophilized enzyme (nominal activity 1,538 U/mg) and diluted with 20 mM HEPES pH 7 to the final concentration. The concentration of the enzyme in the reaction mixture was calculated by comparing the determined activity of the purchased product to the activity of the pure enzyme, reported to be 6,200 U/mg of pure enzyme20, and a molar mass of 545 kDa, corresponding to the homo-hexameric protein. (A) Thermal power observed in the M1 experiment, performed at 25 &#176;C with 2 x 5 &#181;l injections of 20 mM urea (in the injection syringe) into 1 nM JBU (in the sample cell). A spacing of 750 sec between the injections was applied to allow the baseline returning to the initial value. The enthalpy of the reaction is calculated from the integration of each peak and averaging the two values, according to Equation 5. The value is reported in the text. (B) Thermal power observed in the M2 experiment, obtained by titrating 400 mM urea (20 x 5 &#181;l injections) into 15 pM JBU. A spacing of 180 sec was applied between injections, in order to allow the system to reach and maintain the steady state level. dQ/dt at different substrate concentrations is obtained as displacement of the baseline after each addition. The heat of dilution is shown as a blank experiment (gray), performed by titrating substrate into the buffer alone. (C) The baseline displacement in Figure 2B was converted to the reaction rate using Equation 4. Fit of the obtained data (filled squares) was performed using Equation 2, and is represented as a solid line. The value of KM and kcat obtained from the fit are reported in the text.
The total heat change measured in the M1 experiment represents the sum of all events occurring during the reaction under analysis, and depends on the molar enthalpy of all the processes involved, including, if applicable, proton release or uptake from the buffer. In a generic process in which the reacting system acquires protons in a buffered solution, the buffer releases protons, producing additional heat within the reaction cell. Therefore, the measured &#916;H is apparent (&#916;Happ), and includes the intrinsic &#916;H of the reaction (&#916;Hint), as well as the ionization enthalpy of the buffer (&#916;Hion), and ii) the number of exchanging protons (n), according Equation 7:
<img fo:content-width="2.5in" src="/files/ftp_upload/51487/51487eq7.jpg" width="256" /> (7)
Using this equation, in M1 experiments, we can determine n and &#916;Hint by performing the same experiment in buffers with different ionization enthalpies, and plotting the measured &#916;Happ as a function of &#916;Hion specific for the buffer. From the resulting linear regression, n is derived from the slope: a negative slope reflects protons acquired by the buffer, while a positive slope represents protons released from the buffer; &#916;Hint is derived from the intercept on y-axis.
As observed in Equation 6, the overall urea hydrolysis reaction results in the acquisition of a proton H+ from the buffer. Accordingly, as described above, the heat of reaction includes the contribution of buffer deprotonation. In order to calculate the intrinsic &#916;H of the hydrolysis reaction, three M1 experiments were performed in different buffers (HEPES, TrisHCl, phosphate), characterized by three different &#916;Hion. &#916;Hint and the number of proton released from the buffer, calculated from the intercept and the slope of the linear fit of the experimental data (Figure 3) according to Equation 7, were -14.7 kcal/mol&#160;and 0.98 respectively, in full agreement with data previously reported for the same reaction catalyzed by Helicobacter pylori urease7. To our knowledge, no thermodynamic data have been reported for JBU, so far.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51487/51487fig3highres.jpg" width="500" /> 
Figure 3. Determination of &#916;Hint of urease reaction. Values of &#916;Happ in different buffers (filled squares) were determined by performing the M1 experiments in 20 mM HEPES (&#916;Hion = 4.88 kcal/mol), 20 mM TrisHCl (&#916;Hion = 11.34 kcal/mol), and 20 mM phosphate (&#916;Hion = 0.86 kcal/mol)21. Linear regression analysis (solid line) of the data allowed determining &#916;Hint of the reaction, as well as the number of protons exchanged during urea turnover.

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Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

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.

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as ...

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20.5K Views.  University of Bologna. 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.

Video: Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

Retropinacol/Cross-pinacol Coupling Reactions - A Catalytic Access to 1,2-Unsymmetrical Diols

Unsymmetrical 1,2-diols are hardly accessible by reductive pinacol coupling processes. A successful execution of such a transformation is bound to a clear recognition and strict differentiation of two similar carbonyl compounds (aldehydes &#8594; secondary 1,2-diols or ketones &#8594; tertiary 1,2-diols). This fine-tuning is still a challenge and an unsolved problem for an organic chemist. There exist several reports on successful execution of this transformation but they cannot be generalized. Herein we describe a catalytic direct pinacol coupling process which proceeds via a retropinacol/cross-pinacol coupling sequence. Thus, unsymmetrical substituted 1,2-diols can be accessed with almost quantitative yields by means of an&#160;operationally simple performance under very mild conditions. Artificial techniques, such as syringe-pump techniques or delayed additions of reactants are not necessary. The procedure we describe provides a very rapid access to cross-pinacol products (1,2-diols, vicinal diols). A further extension of this new process, e.g. an enantioselective performance could provide a very useful tool for the synthesis of unsymmetrical chiral 1,2-diols.

A novel account for the synthesis of unsymmetrical 1,2-diols based on a retropinacol/cross-pinacol coupling mechanism is described. Due to the catalytic execution of this reaction a considerable improvement compared to conventional cross-pinacol couplings is achieved.

Unsymmetrical 1,2-diols are hardly accessible by reductive pinacol coupling processes. A successful execution of such a transformation is bound to a clear ...

Mizoroki-Heck Cross-coupling Reactions Catalyzed by Dichloro{bis[1,1',1''-(phosphinetriyl)tripiperidine]}palladium Under Mild Reaction Conditions

Dichloro-bis(aminophosphine) complexes of palladium with the general formula of [(P{(NC5H10)3-n(C6H11)n})2Pd(Cl)2] (where n = 0-2), belong to a new family of easy accessible, very cheap, and air stable, but highly active and universally applicable C-C cross-coupling catalysts with an excellent functional group tolerance. Dichloro{bis[1,1',1''-(phosphinetriyl)tripiperidine]}palladium [(P(NC5H10)3)2Pd(Cl)2] (1), the least stable complex within this series towards protons; e.g. in the form of water, allows an eased nanoparticle formation and hence, proved to be the most active Heck catalyst within this series at 100 &#176;C and is a very rare example of an effective and versatile catalyst system that efficiently operates under mild reaction conditions. Rapid and complete catalyst degradation under work-up conditions into phosphonates, piperidinium salts and other, palladium-containing decomposition products assure an easy separation of the coupling products from catalyst and ligands. The facile, cheap, and rapid synthesis of 1,1',1"-(phosphinetriyl)tripiperidine and 1 respectively, the simple and convenient use as well as its excellent catalytic performance in the Heck reaction at 100 &#176;C make 1 to one of the most attractive and greenest Heck catalysts available.
We provide here the visualized protocols for the ligand and catalyst syntheses as well as the reaction protocol for Heck reactions performed at 10 mmol scale at 100 &#176;C and show that this catalyst is suitable for its use in organic syntheses.

Mizoroki-Heck Cross-coupling Reactions Catalyzed by Dichloro{bis[1,1',1''-phosphinetriyltripiperidine]}palladium Under Mild Reaction Conditions

Dichloro{bis[1,1',1''-(phosphinetriyl)tripiperidine]}palladium [(P(NC5H10)3)2Pd(Cl)2] (1) is an easy accessible, cheap, and air stable, but highly active Heck catalyst with an excellent functional group tolerance that efficiently operates under mild reaction conditions to give the coupling products in very high yields.

Dichloro-bis(aminophosphine) complexes of palladium with the general formula of [(P{(NC5H10)3-n(C6H11)n})2Pd(Cl)2] (where n = 0-2), belong to a new family ...

Light-driven Enzymatic Decarboxylation

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. Recycling of the electron donors NADH or NADPH requires the use of additional enzymes and sacrificial substrates. Interestingly, several oxidoreductases accept hydrogen peroxide as electron donor. While being inexpensive, this reagent often reduces the stability of enzymes. A solution to this problem is the in situ generation of the cofactor. The continuous supply of the cofactor at low concentration drives the reaction without impairing enzyme stability. This paper demonstrates a method for the light-catalyzed in situ generation of hydrogen peroxide with the example of the heme-dependent fatty acid decarboxylase OleTJE. The fatty acid decarboxylase OleTJE was discovered due to its unique ability to produce long-chain 1-alkenes from fatty acids, a hitherto unknown enzymatic reaction. 1-alkenes are widely used additives for plasticizers and lubricants. OleTJE has been shown to accept electrons from hydrogen peroxide for the oxidative decarboxylation. While addition of hydrogen peroxide damages the enzyme and results in low yields, in situ generation of the cofactor circumvents this problem. The photobiocatalytic system shows clear advantages regarding enzyme activity and yield, resulting in a simple and efficient system for fatty acid decarboxylation.

We describe a protocol for the light-catalyzed generation of hydrogen peroxide &#8212; a cofactor for oxidative transformations.

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. ...

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

A Protocol for Safe Lithiation Reactions Using Organolithium Reagents

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents warrants proper technique, thorough training, and proper personal protective equipment. To aid in the training of researchers using organolithium reagents, a thorough, step-by-step protocol for the safe and effective use of tert-butyllithium on an inert gas line or within a glovebox is described. As a model reaction, preparation of lithium tert-butyl amide by the reaction of tert-butyl amine with one equivalent of tert-butyl lithium is presented.

The safe and proper use of organolithium reagents is described.

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents ...

Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. Their synthesis by conventional organic chemistry often requires tedious multi-step synthesis processes, with difficult control of the stereochemical outcome. We present a protocol to enzymatically synthetize amino alcohols starting from the readily available L-lysine in 48 h. This protocol combines two chemical reactions that are very difficult to conduct by conventional organic synthesis. In the first step, the regio- and diastereoselective oxidation of an unactivated C-H bond of the lysine side-chain is catalyzed by a dioxygenase; a second regio- and diastereoselective oxidation catalyzed by a regiodivergent dioxygenase can lead to the formation of the 1,2-diols. In the last step, the carboxylic group of the alpha amino acid is cleaved by a pyridoxal-phosphate (PLP) decarboxylase (DC). This decarboxylative step only affects the alpha carbon of the amino acid, retaining the hydroxy-substituted stereogenic center in a beta/gamma position. The resulting amino alcohols are therefore optically enriched. The protocol was successfully applied to the semipreparative-scale synthesis of four amino alcohols. Monitoring of the reactions was conducted by high performance liquid chromatography (HPLC) after derivatization by 1-fluoro-2,4-dinitrobenzene. Straightforward purification by solid-phase extraction (SPE) afforded the amino alcohols with excellent yields (93% to &#62;95%).

Chiral amino alcohols are versatile molecules for use as scaffolds in organic synthesis. Starting from L-lysine, we synthesize amino alcohols by an enzymatic cascade reaction combining diastereoselective C-H oxidation catalyzed by dioxygenase followed by cleavage of the carboxylic acid moiety of the corresponding hydroxyl amino acid by a decarboxylase.

Amino alcohols are versatile compounds with a wide range of applications. For instance, they have been used as chiral scaffolds in organic synthesis. ...

Employing Pressurized Hot Water Extraction (PHWE) to Explore Natural Products Chemistry in the Undergraduate Laboratory

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products research has also found applications as an effective teaching tool. Specifically, this technique has been used to introduce second- and third-year undergraduates to aspects of natural products chemistry in the laboratory. In this report, two experiments are presented: the PHWE of eugenol and acetyleugenol from cloves and the PHWE of seselin and (+)-epoxysuberosin from the endemic Australian plant species Correa reflexa. By employing PHWE in these experiments, the crude clove extract, enriched in eugenol and acetyleugenol, was obtained in 4-9% w/w from cloves by second-year undergraduates and seselin and (+)-epoxysuberosin were isolated in yields of up to 1.1% w/w and 0.9% w/w from C. reflexa by third-year students. The former exercise was developed as a replacement for the traditional steam distillation experiment providing an introduction to extraction and separation techniques, while the latter activity featured guided-inquiry teaching methods in an effort to simulate natural products bioprospecting. This primarily derives from the rapid nature of this PHWE technique relative to traditional extraction methods that are often incompatible with the time constraints associated with undergraduate laboratory experiments. This rapid and practical PHWE method can be used to efficiently isolate various classes of organic molecules from a range of plant species. The complementary nature of this technique relative to more traditional methods has also been demonstrated previously.

Employing Pressurized Hot Water Extraction PHWE to Explore Natural Products Chemistry in the Undergraduate Laboratory

Here, we employ a pressurized hot water extraction (PHWE) method, which utilizes an unmodified household espresso machine to introduce undergraduates to natural products chemistry in the laboratory. Two experiments are presented: PHWE of eugenol and acetyleugenol from cloves and PHWE of seselin and (+)-epoxysuberosin from the Australian plant Correa reflexa.

A recently developed pressurized hot water extraction (PHWE) method which utilizes an unmodified household espresso machine to facilitate natural products ...

Imine Metathesis by Silica-Supported Catalysts Using the Methodology of Surface Organometallic Chemistry

With this protocol, a well-defined singlesite silica-supported heterogeneous catalyst [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] is designed and prepared according to the methodology developed by surface organometallic chemistry (SOMC). In this framework, catalytic cycles can be determined by isolating crucial intermediates. All air-sensitive materials are handled under inert atmosphere (using gloveboxes or a Schlenk line) or high vacuum lines (HVLs, &#60;10-5 mbar). The preparation of SiO2-700 (silica dehydroxylated at 700 &#176;C) and subsequent applications (the grafting of complexes and catalytic runs) requires the use of HVLs and double-Schlenk techniques. Several well-known characterization methods are used, such as Fourier-transform infrared spectroscopy (FTIR), elemental microanalysis, solid-state nuclear magnetic resonance spectroscopy (SSNMR), and state-of-the-art dynamic nuclear polarization surface enhanced NMR spectroscopy (DNP-SENS). FTIR and elemental microanalysis permit scientists to establish the grafting and its stoichiometry. 1H and 13C SSNMR allows the structural determination of the hydrocarbon ligands coordination sphere. DNP SENS is an emerging powerful technique in solid characterization for the detection of poorly sensitive nuclei (15N, in our case). SiO2-700 is treated with about one equivalent of the metal precursor compared to the amount of surface silanol (0.30 mmol&#183;g-1) in pentane at room temperature. Then, volatiles are removed, and the powder samples are dried under dynamic high vacuum to afford the desired materials [(&#8801;Si-O-)Hf(&#951;2&#960;-MeNCH2)(&#951;1-NMe2)(&#951;1-HNMe2)]. After a thermal treatment under high vacuum, the grafted complex is converted into metal imido silica complex [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)]. [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] effectively promotes the metathesis of imines, using the combination of two imine substrates, N-(4-phenylbenzylidene)benzylamine, or N-(4-fluorobenzylidene)-4-fluoroaniline, with N-benzylidenetert-butylamine as substrates. A significantly lower conversion is observed with the blank runs; thus, the presence of the imido group in [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] is correlated to the catalytic performance.

A new group IV metal catalyst for imine metathesis is prepared by grafting amine metal complex onto dehydroxylated silica. Surface metal fragments are characterized using FT-IR, elemental microanalysis, and solid-state NMR spectroscopy. Further dynamic nuclear polarization surface enhanced NMR spectroscopy experiments complement the determination of the coordination sphere.

With this protocol, a well-defined singlesite silica-supported heterogeneous catalyst [(&#8801;Si-O-)Hf(=NMe)(&#951;1-NMe2)] is designed and prepared ...

Enzymatic Synthesis of Epoxidized Metabolites of Docosahexaenoic, Eicosapentaenoic, and Arachidonic Acids

The epoxidized metabolites of various polyunsaturated fatty acids (PUFAs), termed epoxy fatty acids, have a wide range of roles in human physiology. These metabolites are produced endogenously by the cytochrome P450 class of enzymes. Because of their diverse and potent biological effects, there is considerable interest in studying these metabolites. Determining the unique roles of these metabolites in the body is a difficult task, as the epoxy fatty acids must first be obtained in significant amounts and with high purity. Obtaining compounds from natural sources is often labor intensive, and soluble epoxide hydrolases (sEH) rapidly hydrolyze the metabolites. On the other hand, obtaining these metabolites via chemical reactions is very inefficient, due to the difficulty of obtaining pure regioisomers and enantiomers, low yields, and extensive (and expensive) purification. Here, we present an efficient enzymatic synthesis of 19(S),20(R)- and 16(S),17(R)-epoxydocosapentaenoic acids (EDPs) from DHA via epoxidation with BM3, a bacterial CYP450 enzyme isolated originally from Bacillus megaterium (that is readily expressed in Escherichia coli). Characterization and determination of purity is performed with nuclear magnetic resonance spectroscopy (NMR), high-performance liquid chromatography (HPLC), and mass spectrometry (MS). This procedure illustrates the benefits of enzymatic synthesis of PUFA epoxy metabolites, and is applicable to the epoxidation of other fatty acids, including arachidonic acid (AA) and eicosapentaenoic acid (EPA) to produce the analogous epoxyeicosatrienoic acids (EETs) and epoxyeicosatetraenoic acids (EEQs), respectively.

We present a method useful for large-scale enzymatic synthesis and purification of specific enantiomers and regioisomers of epoxides of arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) with the use of a bacterial cytochrome P450 enzyme (BM3).

The epoxidized metabolites of various polyunsaturated fatty acids (PUFAs), termed epoxy fatty acids, have a wide range of roles in human physiology. These ...