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Adaptation of a continuous, calorimetric kinetic assay to study the agmatinase-catalyzed hydrolytic reaction
Analytical Biochemistry 595 (2020) 113618
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Adaptation of a continuous, calorimetric kinetic assay to study the agmatinase-catalyzed hydrolytic reaction
T
Liam A. Wilson, David Garcia, Marcelo Monteiro Pedroso, Benjamin L. Schulz, Luke W. Guddat, Gerhard Schenk∗ School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia
A R T I C LE I N FO
A B S T R A C T
Keywords: Agmatinase Enzyme kinetics Metallohydrolases Ureohydrolase
Ureohydrolases are members of the metallohydrolase family of enzymes. Here, a simple continuous assay for agmatinase (AGM) activity was established by following the degradation of agmatine to urea and putrescine using isothermal titration calorimetry (ITC). ITC is particularly useful for kinetic assays when substrates of interest do not possess suitable chromophores that facilitate the continuous spectrophotometric detection of substrate depletion and/or product formation. In order to assess the accuracy of the ITC-based assay, catalytic parameters were also determined using a discontinuous, colorimetric assay. Both methods resulted in comparable kinetic parameters. From the colorimetric assay the kcat and KM values are 131 s−1 and 0.25 mM, respectively, and from the ITC assay the corresponding parameters are 30 s−1 and 0.45 mM, respectively. The continuous ITC-based assay will facilitate functional studies for an enzyme that is an emerging target for the development of addiction treatments.
1. Introduction Metallohydrolases form a large group of enzymes that require at least one metal ion to catalyze the hydrolysis of a diverse range of phosphate ester and amide bonds [1–3]. Representatives include the Ni2+-dependent ureases, the Zn2+-dependent metallo-β-lactamases and a number of organophosphate-degrading hydrolases that utilize a range of metal ions including Fe2+, Mn2+, Co2+ and even Cd2+ [3–11]. The Mn2+-dependent arginases, agmatinases (AGMs) and the AGM-like protein (ALP) form part of the subgroup of ureohydrolases; in the reactions they catalyze urea is formed as a product. For many metallohydrolases simple spectrophotometric assays are available to measure their catalytic activities; in particular the use of substrates carrying a nitrophenol group has been useful [7,9,12]. For ureohydrolases this is frequently not the case and activities need to be recorded using timeand material-consuming endpoint assays. Here, our focus was on the establishment of a kinetic assay for AGMs and ALPs that is independent of chromophoric properties of the reactants. AGMs and ALPs catalyze the hydrolysis of agmatine (1-amino-4guanidinobutane), a neurotransmitter in the mammalian brain that has been shown to affect a number of neurological disorders through its involvement in polyamine biosynthesis [13,14]. These disorders include alcohol addiction, schizophrenia, anxiety and depression
∗
[13,15–19]. Due to its association with a wide range of neurological pathways, tight regulation of the levels of agmatine in the brain is essential. AGM and ALP play a central role in this regulatory pathway and they have emerged as promising targets for novel addiction and depression treatments [13–18]. Bacterial AGMs and mammalian ALPs have been shown to efficiently degrade agmatine both in vivo and in vitro, and crystal structures of several bacterial AGMs have been reported (Fig. 1) [20–22], but their mode of action is still poorly understood. In vitro enzymatic activity has not yet been demonstrated for mammalian AGMs, but they have been shown to be active in vivo using a complementation assay with a transfected yeast strain [24]. No crystal structure of a mammalian AGM is available but it is evident that these enzymes display significant differences in their active sites when compared amongst each other. While in human AGM the six amino acid ligands of the catalytically essential Mn2+ ions are identical to those in the bacterial AGMs (and the related arginases) only two of them are conserved in mouse AGM (Fig. 1). While such a significant structural variation would be anticipated to result in a large difference in the catalytic efficiency and mechanism of mouse AGM, the limited available data suggest that this enzyme has catalytic properties similar to those of its human counterpart [24]. Even less is known about the structure and mechanism of ALP. Rat brain ALP shares minimal
Corresponding author. E-mail address: [email protected] (G. Schenk).
https://doi.org/10.1016/j.ab.2020.113618 Received 8 January 2020; Received in revised form 5 February 2020; Accepted 9 February 2020 Available online 11 February 2020 0003-2697/ © 2020 Elsevier Inc. All rights reserved.
Analytical Biochemistry 595 (2020) 113618
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competent cells via heat shock. The transformed bacteria were grown in Luria broth at 37 °C in the presence of 50 μg/mL kanamycin for selection. The enzyme was expressed overnight following induction at OD600 = 0.6 using 1 mM IPTG (final concentration). The cells were then collected by centrifugation and resuspended in 25 mM Tris, pH 8.0, 350 mM NaCl with 1 mM MgCl2, 1 mg/mL lysozyme, 1 mg/mL DNase 1 and 1.5 mg/mL EDTA-free protease inhibitor cocktail. Once resuspended the cells were disrupted using sonication. The cells were kept on ice during sonication to avoid overheating. The resulting lysate was then centrifuged and the supernatant was collected and loaded onto a HiPrep IMAC 16/10. High purity AGM was eluted using 150 mM imidazole.
Fig. 1. Active site comparison between E. coli AGM (A) and murine AGM (B). Since the crystal structures of these enzymes have not yet been reported they were modelled using the software “SWISS-MODEL and the structure of the AGM from Deinococcus radiodurans as template [20,23]. The active site of E. coli AGM is very similar to that of the D. radiodurans and human enzymes and can accommodate two Mn2+ ions. Only two of the six metal ligands are conserved in murine AGM (His151 and Asp232), suggesting a significant change in metal binding interactions.
2.2.2. Colorimetric kinetic assay The activity of the purified E. coli AGM was analyzed using the wellestablished colorimetric endpoint assay [25,28]. In short, E. coli AGM was added to a solution of 50 mM glycine, pH 9.0, and 4 mM MnCl2; the final enzyme concentration was 20 nM. The buffer-AGM solution was incubated at 37 °C for 10 min before substrate was added to initiate the catalytic reaction (final reaction volume was 200 μL). The reaction was allowed to run for 1 min before being quenched with 1 mL of a solution containing 23% phosphoric acid and 9% sulphuric acid. 100 μL of 3% α-isonitrosopropiophenone was added and the resulting solution was incubated at 100 °C for 1 h. The colored compound produced by heating the solution is light sensitive, and therefore the boiling was done in amber tubes. Once boiled, the solutions were left to cool to room temperature away from light and the absorbance at 540 nm was measured using a Cary 60 Agilent UV–Vis spectrophotometer. The assays were run in triplicates. In addition to the conditions used above the activity was also tested in 50 mM EPPS, pH 8.5, 1 μM MnCl2 with 42 nM E. coli AGM. Final concentrations of urea were determined with a standard curve of known urea concentrations using the same assay protocol as above.
sequence identity with AGMs (12% and 15% with E. coli and human AGM, respectively), and none of the active site residues characteristic for members of the ureohydrolase family appear conserved [25,26]. Furthermore, ALP activity is autoregulated via the C-terminal LIM domain of the enzyme [27]. It has thus been speculated that AGM and ALP activity both play important roles in regulating the levels of agmatine, in particular in the mammalian brain, but that these enzymes may respond to different stimuli [14]. While detailed functional studies of AGM and ALP are necessary to fully understand their contribution to important neurological functions, the lack of a simple and robust kinetic assay has complicated progress in this area. Neither the substrate agmatine, nor the reaction products urea and putrescine have suitable chromophoric properties that enable a continuous spectrophotometric assay. Consequently, a discontinuous assay is routinely employed that relies on the reaction between urea and the dye α-isonitrosopropiophenone to form a compound that is visible in the UV–visible spectrum (λmax = 540 nm) [21,28]. This assay is expensive both in terms of time and materials consumed, and renders measurements of kinetic parameters over a wide range of conditions cumbersome. In order to address this current limitation we have adapted a previously established continuous activity assay that relies on the use of isothermal titration calorimetry (ITC) and implemented it to effectively monitor the agmatinase reaction [29,30]. This method is independent on chromophoric properties of the reactants and since the method relies on monitoring heat changes associated with the hydrolysis of the substrate it can easily be adapted for other reaction conditions and hydrolase enzymes.
2.2.3. Theoretical background for the ITC-based activity assay ITC is a convenient method to study binding interactions between ligands, but its potential in kinetic assays has not yet been thoroughly exploited. Motivated by initial studies which have effectively applied this assay with enzymes such as xylanases, ureases from Helicobacter pylori, Sporosarcina pasteurii and Canavalia ensiformis and the phosphodiesterases Rv0805 and GpdQ, we have adapted and applied this assay to monitor agmatine hydrolysis by ITC [29–33]. Kinetic parameters can be determined through a combination of two alternative ITC measurements, one based on a single injection and one on multiple injections, which have been previously described in detail [29,30]. Briefly, the single injection measurement involves a single injection of a known amount of substrate into a solution containing an excess of enzyme. The change in enthalpy of the system is then monitored until the reaction has run to completion and the change in enthalpy returns to baseline. From this the molar enthalpy of the reaction (defined as ΔHapp in kJ/ mol) can be determined by the integration of the enthalpy change over time of a known amount of substrate. This molar enthalpy can then be used to convert the rate of the change of enthalpy measured in the multiple injection method to a molar rate [29–31]. The multiple injection method, involves subsequent injections made once the enthalpic response from the previous injection had reached its peak, preventing the enthalpic response from returning to the baseline. This results in a steady state rate of enthalpy change corresponding to the rate of the reaction at the substrate concentration present in the reaction cell after each injection which can be converted into a molar rate (and subsequently used to generate a Michaelis-Menten curve and determine the kinetic parameters) using the molar enthalpy. This conversion is done using Equation (1):
2. Materials and methods 2.1. Materials E. coli BL21 (DE3) host cells were purchased from Novagen. The pET-24a (+) plasmid containing the E. coli AGM gene was purchased from General Biosystems Inc. The AGM gene used was from E. coli K-12 although this sequence is highly conserved among all strains of E. coli in the NCBI database. All purification equipment (both chromatography columns and fast liquid chromatography systems) were purchased from GE Healthcare. All chemicals and buffers were purchased from Sigma Chemical Company. All ITC assays were run using a NanoITC (TA Instruments). 2.2. Methods 2.2.1. Protein expression and purification E. coli AGM was used as the model system to establish the ITC-based assay to measure the rate of agmatine hydrolysis. The open reading frame encoding E. coli AGM was cloned into pET-24a (+), providing a C-terminal His-Tag, and was transformed into E. coli BL21 (DE3)
Rate =
2
1 dQ × V × ΔHapp dt
(1)
Analytical Biochemistry 595 (2020) 113618
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the enthalpy. Consequently, ΔHapp recorded for the reaction in glycine buffer was used for all calculations. Experimental data from multiple injection measurements, including that of a blank, are shown in Fig. 2B, and the resulting MichaelisMenten curve is shown in Fig. 2C. The relevant kinetic parameters (kcat and KM) for the E. coli AGM agmatinase reaction were determined using the ITC and the colorimetric assay in two different assay conditions (summarized in Table 1). Note that the magnitude of the KM value is of the same order as the lowest concentration of substrate that could be added to obtain reliable and reproducible results. However, as shown in Fig. 2C the curvature of the data and the low error associated with individual data points allow for an accurate estimate of the KM value.
where V is the volume of the reaction, and dQ/dt is the steady state enthalpy change of the system. In reactions in which a proton is produced the enthalpic effect of the protonation of the buffer present needs to be taken into account when measuring a reaction in multiple buffer systems as it can affect the value of ΔHapp. This affect is due to the heat which is released by the ionization event (ΔHion), and which is unique for each buffer system. Consequently, if a reaction is run in two different buffer systems (e.g. glycine and EPPS) different ΔHapp values may need to be taken into account [29]. 2.2.4. ITC kinetic assay method The reaction cell was filled either with 10 μM MnCl2 and 60 nM AGM or 4 mM Mn2+ and 3 μM AGM, both in 50 mM glycine, pH 9.0, for the multiple and single injection measurements, respectively (final reaction volumes were 400 μL). The assays were also run in 50 mM EPPS, pH 8.5, 1 μM MnCl2 with 60 nM AGM. All assays were run at 37 °C with a stirring speed of 300 rpm. The reference cell was filled with the corresponding buffer-MnCl2 solution without AGM present. Note that Mn2+ was added to all assays to reproduce previous studies that demonstrated that a stable AGM activity requires the presence of added metal ions in the buffer [21]. Before each experiment, the enzymebuffer solution was equilibrated at 37 °C. Single injection measurements were run by injecting 2 μL of 200 mM agmatine (final concentration of 1 mM) into the buffer-enzyme solution. Three separate injections were made with a spacing of 900 s. Multiple injection measurements were performed by 15 injections of 1 μL of a 100 mM agmatine solution (final concentration of 0.25 mM per injection) into the buffer-enzyme mixture, with a spacing of 110 s. The assays were run in duplicates. The raw ITC data were processed using the NanoAnalyze software from TA instruments.
4. Discussion The development of a simple, reliable and rapid assay to measure the rate of agmatine hydrolysis will facilitate detailed investigations into the regulation and metabolic functions of AGM and ALP. E. coli AGM was a convenient model system to optimize the assay. The activity of E. coli AGM measured by ITC in the presence of 4 mM Mn2+ (the standard conditions for the colorimetric assay) was not significantly higher than the blank. One potential reason for this is an endothermic interaction between the Mn2+ and one of the other reagents present. Activities could only be measured reliably when the concentration of Mn2+ was reduced significantly (Table 1). This highlights a potential issue, when observing the reactions of certain enzymes, which may require high concentrations of different reactants. If it is necessary for a particular reaction to have high concentration of a cofactor or other additive, the addition of which is associated with a significant change in enthalpy, then this may cause inaccuracies in the measurement of the enthalpy change due to the reaction of interest. Although this may cause issues when observing some reactions, in most cases, this can be accounted for by running appropriate controls. Conversely the colorimetric assay has been shown to be affected by the presence of ureido compounds in biological samples [37]. While the effect of these compounds was shown to be removed by purification of the sample with ion exchange chromatography the efficacy of removing these interfering compounds with other forms of chromatography (e.g. metal affinity) is unknown [37]. It should also be pointed out that the indirect nature of the colorimetric assay may cause experimental errors, including inherent delays in the color development. Under conditions with reduced Mn2+, to avoid competing enthalpic interactions, the KM measured in the ITC experiment is in good agreement with that reported using the colorimetric endpoint assay (see Table 1). The kcat value recorded in the ITC experiment is four-fold smaller than the corresponding value obtained in the colorimetric experiment. While this may be due to partial occupancy of the active site with Mn2+ in the ITC-based assay (due to the low Mn2+ concentration in the experiment) we note that the reported values for the KM values in the literature also vary by a factor of ~4 relative to the experimental KM. Thus, the observed variation of the rates measured with the ITC and colorimetric assays lies within the margin of error of different measurements. Nonetheless, in order to determine whether the low Mn2+ concentration or buffer effects present in the ITC assay were major contributors to the difference in kcat values the ITC and colorimetric assays were repeated in a different buffer system with reduced Mn2+ concentrations (i.e. EPPS buffer systems; Table 1). In both assays the Mn2+ concentration was maintained at 1 μM. Under these conditions the kcat values in both experiments were reduced ~ three-fold when compared to the assays in the glycine buffer system. The KM value is not greatly affected in the colorimetric assay (0.25 mM vs 0.20 mM; Table 1) but reduced four-fold in the ITC-based assay (0.45 mM vs 0.12 mM). Overall, these variations indicate that there is no clear trend or correlation between the changes of the assay condition and the catalytic parameters, as demonstrated by the catalytic efficiency (kcat/ KM ratio). This suggests that the observed variations between the ITC
3. Results 3.1. Enzyme purification and activity E. coli AGM was selected as a model system to establish the experimental framework for an ITC-based activity assay because it is the best-studied AGM, can easily be purified to homogeneity, is stable and has a high catalytic rate [21,34,35]. An average purification of Cterminal hexa-histidine-tagged AGM yields approximately 40 mg per liter of growth medium following a simple 1-step purification protocol using a Ni2+- IMAC column. In order to compare E. coli AGM prepared here with previous preparations, and to establish a benchmark value for the catalytic parameters obtained from the ITC-based method we employed the colorimetric endpoint assay to determine the kcat and KM values of the enzyme [21,35,36]. The values determined for kcat and KM are 131 ± 3 s−1 and 0.25 ± 0.02 mM. Reported literature values measured under identical conditions are kcat = 120 ± 10 s−1, KM = 1.1 ± 0.2 mM [36]; while the rates are in excellent agreement the KM values vary ~ four-fold. 3.2. ITC assays When monitoring the AGM reaction with the E. coli enzyme a considerable exothermic heat change is observed in the single injection experiment (Fig. 2A). While the heat of dilution of the substrate (shown in red in Fig. 2A) is measurable, it is small in comparison to the overall heat of the reaction. From the thermograms the molar enthalpy ΔHapp for the AGM-catalyzed hydrolysis of agmatine in 50 mM glycine, pH 9.0 with 4 mM Mn2+ is determined to be −20.3 ± 0.2 kJ/mol. The ΔHapp was also determined for the reaction in 50 mM EPPS, pH 8.5, in the presence of 1 μM Mn2+ in order to account for potential changes in ΔHion between the buffers. However, ΔHapp in EPPS = −20.4 ± 0.06 kJ/mol, demonstrating that the buffer has no significant effect on 3
Analytical Biochemistry 595 (2020) 113618
L.A. Wilson, et al.
Fig. 2. A) Single injection method showing the hydrolysis of 2 μL injections of 200 mM agmatine by 3 μM E. coli AGM in 50 mM glycine, pH 9.0, and 4 mM Mn2+ (shown in blue). Controls were also run using the same conditions without enzyme in the reaction cell (shown in red) and with water in the injection syringe (shown in green). Data were collected in triplicates to show reproducibility and to test for product inhibition. B) Multiple injection measurements showing the hydrolysis of 1 μL injections of 100 mM agmatine by 60 nM E. coli AGM in 50 mM glycine, pH 9.0, 10 μM Mn2+. Injections of substrate were made every 110 s preventing the enthalpic response from returning to the baseline. C) Michaelis-Menten curve generated from the multiple injection method and the molar enthalpy determined in the single injection method. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Table 1 Comparison of the kinetic constants of the E. coli AGM reaction as determined by the colorimetric and the ITC assays under different conditions. The kinetic constants recorded in glycine buffer, pH 9.0, were obtained using 4 mM Mn2+ for the colorimetric assay and 10 μM Mn2+ for the ITC assay. The corresponding parameters were also measured using the EPPS buffer system, pH 8.5, in the presence of 1 μM Mn2+ for both assays. Conditions
Assay
kcat (s−1)
KM (mM)
kcat/KM (s−1mM−1)
Glycine (pH 9.0)
4 mM Mn2+ 10 μM Mn2+
Colorimetric ITC
131 ± 3 30 ± 0.5
0.25 ± 0.02 0.45 ± 0.03
516 67
EPPS (pH 8.5)
1 μM Mn2+ 1 μM Mn2+
Colorimetric ITC
43 ± 2 10.8 ± 0.1
0.20 ± 0.03 0.12 ± 0.01
215 90
and colorimetric assays are not linked to differences in experimental conditions (i.e. Mn2+ concentration) but are inherent to the methods themselves. However, the overall agreement between the two methods is reasonable.
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5. Conclusion The availability of an efficient and continuous kinetic assay that is able to monitor the AGM-catalyzed reaction will enable enhanced research into enzymes with AGM activity in particular and ureahydrolases in general. The ITC-based assay developed here is practical as it is a rapid and efficient alternative to the colorimetric assay. It is also based on an automated system of timed injection and measurement. Thus, experimental errors introduced into the measurement by a laborintensive method is minimized. Funding This research was supported by the Australian Research Council (DP150104358). DG also acknowledges the Comisión Nacional de Investigación Científica y Tecnológica – CONICYT, Chile, for their support in form of a BECAS postdoctoral fellowship. CRediT authorship contribution statement Liam A. Wilson: Investigation, Writing - original draft. David Garcia: Methodology, Writing - review & editing. Marcelo Monteiro Pedroso: Writing - review & editing, Methodology. Benjamin L. Schulz: Conceptualization, Project administration, Supervision. Luke W. Guddat: Project administration, Supervision. Gerhard Schenk: Project administration, Writing - review & editing, Supervision. References [1] D.E. Wilcox, Binuclear metallohydrolases, Chem. Rev. 96 (1996) 2435–2458, https://doi.org/10.1021/cr950043b.
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M. Garcia, V. Lopez, G. Schenk, E. Uribe, Insight on the interaction of an agmatinase-like protein with Mn2+ activator ions, J. Inorg. Biochem. 145 (2015) 65–69, https://doi.org/10.1016/j.jinorgbio.2015.01.008. V. Castro, P. Fuentealba, A. Henriquez, A. Vallejos, J. Benitez, M. Lobos, B. Diaz, N. Carvajal, E. Uribe, Evidence for an inhibitory LIM domain in a rat brain agmatinase-like protein, Arch. Biochem. Biophys. 512 (2011) 107–110, https://doi.org/ 10.1016/j.abb.2011.05.003. R.M. Archibald, Colorimetric determination of urea, J. Biol. Chem. 157 (1945) 507–518. M.J. Todd, J. Gomez, Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? Anal. Biochem. 296 (2001) 179–187, https://doi.org/10. 1006/abio.2001.5218. M.M. Pedroso, F. Ely, T. Lonhienne, L.R. Gahan, D.L. Ollis, L.W. Guddat, G. Schenk, Determination of the catalytic activity of binuclear metallohydrolases using isothermal titration calorimetry, J. Biol. Inorg. Chem. 19 (2014) 389–398, https://doi. org/10.1007/s00775-013-1079-0. M.J. Baumann, L. Murphy, N. Lei, K.B.R.M. Krogh, K. Borch, P. Westh, Advantages of isothermal titration calorimetry for xylanase kinetics in comparison to chemicalreducing-end assays, Anal. Biochem. 410 (2011) 19–26, https://doi.org/10.1016/j. ab.2010.11.001. S. Benini, M. Cianci, L. Mazzei, S. Ciurli, Fluoride inhibition of Sporosarcina pasteurii urease: structure and thermodynamics, J. Biol. Inorg. Chem. 19 (2014) 1243–1261, https://doi.org/10.1007/s00775-014-1182-x. L. Mazzei, M. Cianci, S. Benini, L. Bertini, F. Musiani, S. Ciurli, Kinetic and structural studies reveal a unique binding mode of sulfite to the nickel center in urease, J. Inorg. Biochem. 154 (2016) 42–49, https://doi.org/10.1016/j.jinorgbio.2015.11. 003. M. Salas, V. López, E. Uribe, N. Carvajal, Studies on the interaction of Escherichia coli agmatinase with manganese ions: structural and kinetic studies of the H126N and H151N variants, J. Inorg. Biochem. 98 (2004) 1032–1036, https://doi.org/10. 1016/j.jinorgbio.2004.02.022. M. Salas, R. Rodríguez, N. López, E. Uribe, V. Lopez, N. Carvajal, Insights into the reaction mechanism of Escherichia coli agmatinase by site-directed mutagenesis and molecular modelling: a critical role for aspartate 153, Eur. J. Biochem. 269 (2002) 5522–5526, https://doi.org/10.1046/j.1432-1033.2002.03255.x. N. Carvajal, M.a.S. Orellana, M. Salas, P. Enrı́quez, R. Alarcón, E. Uribe, V. López, Kinetic studies and site-directed mutagenesis of Escherichia coli agmatinase. A role for Glu274 in binding and correct positioning of the substrate guanidinium group, Arch. Biochem. Biophys. 430 (2004) 185–190, https://doi.org/10.1016/j.abb. 2004.07.005. B.J. Shelp, K. Sieciechowicz, R.J. Ireland, K.W. Joy, Determination of urea and ammonia in leaf extracts: application to ureide metabolism, Can. J. Bot. 63 (1985) 1135–1140, https://doi.org/10.1139/b85-156.
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Adaptation of a continuous, calorimetric kinetic assay to study the agmatinase-catalyzed hydrolytic reaction
T
Liam A. Wilson, David Garcia, Marcelo Monteiro Pedroso, Benjamin L. Schulz, Luke W. Guddat, Gerhard Schenk∗ School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia
A R T I C LE I N FO
A B S T R A C T
Keywords: Agmatinase Enzyme kinetics Metallohydrolases Ureohydrolase
Ureohydrolases are members of the metallohydrolase family of enzymes. Here, a simple continuous assay for agmatinase (AGM) activity was established by following the degradation of agmatine to urea and putrescine using isothermal titration calorimetry (ITC). ITC is particularly useful for kinetic assays when substrates of interest do not possess suitable chromophores that facilitate the continuous spectrophotometric detection of substrate depletion and/or product formation. In order to assess the accuracy of the ITC-based assay, catalytic parameters were also determined using a discontinuous, colorimetric assay. Both methods resulted in comparable kinetic parameters. From the colorimetric assay the kcat and KM values are 131 s−1 and 0.25 mM, respectively, and from the ITC assay the corresponding parameters are 30 s−1 and 0.45 mM, respectively. The continuous ITC-based assay will facilitate functional studies for an enzyme that is an emerging target for the development of addiction treatments.
1. Introduction Metallohydrolases form a large group of enzymes that require at least one metal ion to catalyze the hydrolysis of a diverse range of phosphate ester and amide bonds [1–3]. Representatives include the Ni2+-dependent ureases, the Zn2+-dependent metallo-β-lactamases and a number of organophosphate-degrading hydrolases that utilize a range of metal ions including Fe2+, Mn2+, Co2+ and even Cd2+ [3–11]. The Mn2+-dependent arginases, agmatinases (AGMs) and the AGM-like protein (ALP) form part of the subgroup of ureohydrolases; in the reactions they catalyze urea is formed as a product. For many metallohydrolases simple spectrophotometric assays are available to measure their catalytic activities; in particular the use of substrates carrying a nitrophenol group has been useful [7,9,12]. For ureohydrolases this is frequently not the case and activities need to be recorded using timeand material-consuming endpoint assays. Here, our focus was on the establishment of a kinetic assay for AGMs and ALPs that is independent of chromophoric properties of the reactants. AGMs and ALPs catalyze the hydrolysis of agmatine (1-amino-4guanidinobutane), a neurotransmitter in the mammalian brain that has been shown to affect a number of neurological disorders through its involvement in polyamine biosynthesis [13,14]. These disorders include alcohol addiction, schizophrenia, anxiety and depression
∗
[13,15–19]. Due to its association with a wide range of neurological pathways, tight regulation of the levels of agmatine in the brain is essential. AGM and ALP play a central role in this regulatory pathway and they have emerged as promising targets for novel addiction and depression treatments [13–18]. Bacterial AGMs and mammalian ALPs have been shown to efficiently degrade agmatine both in vivo and in vitro, and crystal structures of several bacterial AGMs have been reported (Fig. 1) [20–22], but their mode of action is still poorly understood. In vitro enzymatic activity has not yet been demonstrated for mammalian AGMs, but they have been shown to be active in vivo using a complementation assay with a transfected yeast strain [24]. No crystal structure of a mammalian AGM is available but it is evident that these enzymes display significant differences in their active sites when compared amongst each other. While in human AGM the six amino acid ligands of the catalytically essential Mn2+ ions are identical to those in the bacterial AGMs (and the related arginases) only two of them are conserved in mouse AGM (Fig. 1). While such a significant structural variation would be anticipated to result in a large difference in the catalytic efficiency and mechanism of mouse AGM, the limited available data suggest that this enzyme has catalytic properties similar to those of its human counterpart [24]. Even less is known about the structure and mechanism of ALP. Rat brain ALP shares minimal
Corresponding author. E-mail address: [email protected] (G. Schenk).
https://doi.org/10.1016/j.ab.2020.113618 Received 8 January 2020; Received in revised form 5 February 2020; Accepted 9 February 2020 Available online 11 February 2020 0003-2697/ © 2020 Elsevier Inc. All rights reserved.
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competent cells via heat shock. The transformed bacteria were grown in Luria broth at 37 °C in the presence of 50 μg/mL kanamycin for selection. The enzyme was expressed overnight following induction at OD600 = 0.6 using 1 mM IPTG (final concentration). The cells were then collected by centrifugation and resuspended in 25 mM Tris, pH 8.0, 350 mM NaCl with 1 mM MgCl2, 1 mg/mL lysozyme, 1 mg/mL DNase 1 and 1.5 mg/mL EDTA-free protease inhibitor cocktail. Once resuspended the cells were disrupted using sonication. The cells were kept on ice during sonication to avoid overheating. The resulting lysate was then centrifuged and the supernatant was collected and loaded onto a HiPrep IMAC 16/10. High purity AGM was eluted using 150 mM imidazole.
Fig. 1. Active site comparison between E. coli AGM (A) and murine AGM (B). Since the crystal structures of these enzymes have not yet been reported they were modelled using the software “SWISS-MODEL and the structure of the AGM from Deinococcus radiodurans as template [20,23]. The active site of E. coli AGM is very similar to that of the D. radiodurans and human enzymes and can accommodate two Mn2+ ions. Only two of the six metal ligands are conserved in murine AGM (His151 and Asp232), suggesting a significant change in metal binding interactions.
2.2.2. Colorimetric kinetic assay The activity of the purified E. coli AGM was analyzed using the wellestablished colorimetric endpoint assay [25,28]. In short, E. coli AGM was added to a solution of 50 mM glycine, pH 9.0, and 4 mM MnCl2; the final enzyme concentration was 20 nM. The buffer-AGM solution was incubated at 37 °C for 10 min before substrate was added to initiate the catalytic reaction (final reaction volume was 200 μL). The reaction was allowed to run for 1 min before being quenched with 1 mL of a solution containing 23% phosphoric acid and 9% sulphuric acid. 100 μL of 3% α-isonitrosopropiophenone was added and the resulting solution was incubated at 100 °C for 1 h. The colored compound produced by heating the solution is light sensitive, and therefore the boiling was done in amber tubes. Once boiled, the solutions were left to cool to room temperature away from light and the absorbance at 540 nm was measured using a Cary 60 Agilent UV–Vis spectrophotometer. The assays were run in triplicates. In addition to the conditions used above the activity was also tested in 50 mM EPPS, pH 8.5, 1 μM MnCl2 with 42 nM E. coli AGM. Final concentrations of urea were determined with a standard curve of known urea concentrations using the same assay protocol as above.
sequence identity with AGMs (12% and 15% with E. coli and human AGM, respectively), and none of the active site residues characteristic for members of the ureohydrolase family appear conserved [25,26]. Furthermore, ALP activity is autoregulated via the C-terminal LIM domain of the enzyme [27]. It has thus been speculated that AGM and ALP activity both play important roles in regulating the levels of agmatine, in particular in the mammalian brain, but that these enzymes may respond to different stimuli [14]. While detailed functional studies of AGM and ALP are necessary to fully understand their contribution to important neurological functions, the lack of a simple and robust kinetic assay has complicated progress in this area. Neither the substrate agmatine, nor the reaction products urea and putrescine have suitable chromophoric properties that enable a continuous spectrophotometric assay. Consequently, a discontinuous assay is routinely employed that relies on the reaction between urea and the dye α-isonitrosopropiophenone to form a compound that is visible in the UV–visible spectrum (λmax = 540 nm) [21,28]. This assay is expensive both in terms of time and materials consumed, and renders measurements of kinetic parameters over a wide range of conditions cumbersome. In order to address this current limitation we have adapted a previously established continuous activity assay that relies on the use of isothermal titration calorimetry (ITC) and implemented it to effectively monitor the agmatinase reaction [29,30]. This method is independent on chromophoric properties of the reactants and since the method relies on monitoring heat changes associated with the hydrolysis of the substrate it can easily be adapted for other reaction conditions and hydrolase enzymes.
2.2.3. Theoretical background for the ITC-based activity assay ITC is a convenient method to study binding interactions between ligands, but its potential in kinetic assays has not yet been thoroughly exploited. Motivated by initial studies which have effectively applied this assay with enzymes such as xylanases, ureases from Helicobacter pylori, Sporosarcina pasteurii and Canavalia ensiformis and the phosphodiesterases Rv0805 and GpdQ, we have adapted and applied this assay to monitor agmatine hydrolysis by ITC [29–33]. Kinetic parameters can be determined through a combination of two alternative ITC measurements, one based on a single injection and one on multiple injections, which have been previously described in detail [29,30]. Briefly, the single injection measurement involves a single injection of a known amount of substrate into a solution containing an excess of enzyme. The change in enthalpy of the system is then monitored until the reaction has run to completion and the change in enthalpy returns to baseline. From this the molar enthalpy of the reaction (defined as ΔHapp in kJ/ mol) can be determined by the integration of the enthalpy change over time of a known amount of substrate. This molar enthalpy can then be used to convert the rate of the change of enthalpy measured in the multiple injection method to a molar rate [29–31]. The multiple injection method, involves subsequent injections made once the enthalpic response from the previous injection had reached its peak, preventing the enthalpic response from returning to the baseline. This results in a steady state rate of enthalpy change corresponding to the rate of the reaction at the substrate concentration present in the reaction cell after each injection which can be converted into a molar rate (and subsequently used to generate a Michaelis-Menten curve and determine the kinetic parameters) using the molar enthalpy. This conversion is done using Equation (1):
2. Materials and methods 2.1. Materials E. coli BL21 (DE3) host cells were purchased from Novagen. The pET-24a (+) plasmid containing the E. coli AGM gene was purchased from General Biosystems Inc. The AGM gene used was from E. coli K-12 although this sequence is highly conserved among all strains of E. coli in the NCBI database. All purification equipment (both chromatography columns and fast liquid chromatography systems) were purchased from GE Healthcare. All chemicals and buffers were purchased from Sigma Chemical Company. All ITC assays were run using a NanoITC (TA Instruments). 2.2. Methods 2.2.1. Protein expression and purification E. coli AGM was used as the model system to establish the ITC-based assay to measure the rate of agmatine hydrolysis. The open reading frame encoding E. coli AGM was cloned into pET-24a (+), providing a C-terminal His-Tag, and was transformed into E. coli BL21 (DE3)
Rate =
2
1 dQ × V × ΔHapp dt
(1)
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the enthalpy. Consequently, ΔHapp recorded for the reaction in glycine buffer was used for all calculations. Experimental data from multiple injection measurements, including that of a blank, are shown in Fig. 2B, and the resulting MichaelisMenten curve is shown in Fig. 2C. The relevant kinetic parameters (kcat and KM) for the E. coli AGM agmatinase reaction were determined using the ITC and the colorimetric assay in two different assay conditions (summarized in Table 1). Note that the magnitude of the KM value is of the same order as the lowest concentration of substrate that could be added to obtain reliable and reproducible results. However, as shown in Fig. 2C the curvature of the data and the low error associated with individual data points allow for an accurate estimate of the KM value.
where V is the volume of the reaction, and dQ/dt is the steady state enthalpy change of the system. In reactions in which a proton is produced the enthalpic effect of the protonation of the buffer present needs to be taken into account when measuring a reaction in multiple buffer systems as it can affect the value of ΔHapp. This affect is due to the heat which is released by the ionization event (ΔHion), and which is unique for each buffer system. Consequently, if a reaction is run in two different buffer systems (e.g. glycine and EPPS) different ΔHapp values may need to be taken into account [29]. 2.2.4. ITC kinetic assay method The reaction cell was filled either with 10 μM MnCl2 and 60 nM AGM or 4 mM Mn2+ and 3 μM AGM, both in 50 mM glycine, pH 9.0, for the multiple and single injection measurements, respectively (final reaction volumes were 400 μL). The assays were also run in 50 mM EPPS, pH 8.5, 1 μM MnCl2 with 60 nM AGM. All assays were run at 37 °C with a stirring speed of 300 rpm. The reference cell was filled with the corresponding buffer-MnCl2 solution without AGM present. Note that Mn2+ was added to all assays to reproduce previous studies that demonstrated that a stable AGM activity requires the presence of added metal ions in the buffer [21]. Before each experiment, the enzymebuffer solution was equilibrated at 37 °C. Single injection measurements were run by injecting 2 μL of 200 mM agmatine (final concentration of 1 mM) into the buffer-enzyme solution. Three separate injections were made with a spacing of 900 s. Multiple injection measurements were performed by 15 injections of 1 μL of a 100 mM agmatine solution (final concentration of 0.25 mM per injection) into the buffer-enzyme mixture, with a spacing of 110 s. The assays were run in duplicates. The raw ITC data were processed using the NanoAnalyze software from TA instruments.
4. Discussion The development of a simple, reliable and rapid assay to measure the rate of agmatine hydrolysis will facilitate detailed investigations into the regulation and metabolic functions of AGM and ALP. E. coli AGM was a convenient model system to optimize the assay. The activity of E. coli AGM measured by ITC in the presence of 4 mM Mn2+ (the standard conditions for the colorimetric assay) was not significantly higher than the blank. One potential reason for this is an endothermic interaction between the Mn2+ and one of the other reagents present. Activities could only be measured reliably when the concentration of Mn2+ was reduced significantly (Table 1). This highlights a potential issue, when observing the reactions of certain enzymes, which may require high concentrations of different reactants. If it is necessary for a particular reaction to have high concentration of a cofactor or other additive, the addition of which is associated with a significant change in enthalpy, then this may cause inaccuracies in the measurement of the enthalpy change due to the reaction of interest. Although this may cause issues when observing some reactions, in most cases, this can be accounted for by running appropriate controls. Conversely the colorimetric assay has been shown to be affected by the presence of ureido compounds in biological samples [37]. While the effect of these compounds was shown to be removed by purification of the sample with ion exchange chromatography the efficacy of removing these interfering compounds with other forms of chromatography (e.g. metal affinity) is unknown [37]. It should also be pointed out that the indirect nature of the colorimetric assay may cause experimental errors, including inherent delays in the color development. Under conditions with reduced Mn2+, to avoid competing enthalpic interactions, the KM measured in the ITC experiment is in good agreement with that reported using the colorimetric endpoint assay (see Table 1). The kcat value recorded in the ITC experiment is four-fold smaller than the corresponding value obtained in the colorimetric experiment. While this may be due to partial occupancy of the active site with Mn2+ in the ITC-based assay (due to the low Mn2+ concentration in the experiment) we note that the reported values for the KM values in the literature also vary by a factor of ~4 relative to the experimental KM. Thus, the observed variation of the rates measured with the ITC and colorimetric assays lies within the margin of error of different measurements. Nonetheless, in order to determine whether the low Mn2+ concentration or buffer effects present in the ITC assay were major contributors to the difference in kcat values the ITC and colorimetric assays were repeated in a different buffer system with reduced Mn2+ concentrations (i.e. EPPS buffer systems; Table 1). In both assays the Mn2+ concentration was maintained at 1 μM. Under these conditions the kcat values in both experiments were reduced ~ three-fold when compared to the assays in the glycine buffer system. The KM value is not greatly affected in the colorimetric assay (0.25 mM vs 0.20 mM; Table 1) but reduced four-fold in the ITC-based assay (0.45 mM vs 0.12 mM). Overall, these variations indicate that there is no clear trend or correlation between the changes of the assay condition and the catalytic parameters, as demonstrated by the catalytic efficiency (kcat/ KM ratio). This suggests that the observed variations between the ITC
3. Results 3.1. Enzyme purification and activity E. coli AGM was selected as a model system to establish the experimental framework for an ITC-based activity assay because it is the best-studied AGM, can easily be purified to homogeneity, is stable and has a high catalytic rate [21,34,35]. An average purification of Cterminal hexa-histidine-tagged AGM yields approximately 40 mg per liter of growth medium following a simple 1-step purification protocol using a Ni2+- IMAC column. In order to compare E. coli AGM prepared here with previous preparations, and to establish a benchmark value for the catalytic parameters obtained from the ITC-based method we employed the colorimetric endpoint assay to determine the kcat and KM values of the enzyme [21,35,36]. The values determined for kcat and KM are 131 ± 3 s−1 and 0.25 ± 0.02 mM. Reported literature values measured under identical conditions are kcat = 120 ± 10 s−1, KM = 1.1 ± 0.2 mM [36]; while the rates are in excellent agreement the KM values vary ~ four-fold. 3.2. ITC assays When monitoring the AGM reaction with the E. coli enzyme a considerable exothermic heat change is observed in the single injection experiment (Fig. 2A). While the heat of dilution of the substrate (shown in red in Fig. 2A) is measurable, it is small in comparison to the overall heat of the reaction. From the thermograms the molar enthalpy ΔHapp for the AGM-catalyzed hydrolysis of agmatine in 50 mM glycine, pH 9.0 with 4 mM Mn2+ is determined to be −20.3 ± 0.2 kJ/mol. The ΔHapp was also determined for the reaction in 50 mM EPPS, pH 8.5, in the presence of 1 μM Mn2+ in order to account for potential changes in ΔHion between the buffers. However, ΔHapp in EPPS = −20.4 ± 0.06 kJ/mol, demonstrating that the buffer has no significant effect on 3
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Fig. 2. A) Single injection method showing the hydrolysis of 2 μL injections of 200 mM agmatine by 3 μM E. coli AGM in 50 mM glycine, pH 9.0, and 4 mM Mn2+ (shown in blue). Controls were also run using the same conditions without enzyme in the reaction cell (shown in red) and with water in the injection syringe (shown in green). Data were collected in triplicates to show reproducibility and to test for product inhibition. B) Multiple injection measurements showing the hydrolysis of 1 μL injections of 100 mM agmatine by 60 nM E. coli AGM in 50 mM glycine, pH 9.0, 10 μM Mn2+. Injections of substrate were made every 110 s preventing the enthalpic response from returning to the baseline. C) Michaelis-Menten curve generated from the multiple injection method and the molar enthalpy determined in the single injection method. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Table 1 Comparison of the kinetic constants of the E. coli AGM reaction as determined by the colorimetric and the ITC assays under different conditions. The kinetic constants recorded in glycine buffer, pH 9.0, were obtained using 4 mM Mn2+ for the colorimetric assay and 10 μM Mn2+ for the ITC assay. The corresponding parameters were also measured using the EPPS buffer system, pH 8.5, in the presence of 1 μM Mn2+ for both assays. Conditions
Assay
kcat (s−1)
KM (mM)
kcat/KM (s−1mM−1)
Glycine (pH 9.0)
4 mM Mn2+ 10 μM Mn2+
Colorimetric ITC
131 ± 3 30 ± 0.5
0.25 ± 0.02 0.45 ± 0.03
516 67
EPPS (pH 8.5)
1 μM Mn2+ 1 μM Mn2+
Colorimetric ITC
43 ± 2 10.8 ± 0.1
0.20 ± 0.03 0.12 ± 0.01
215 90
and colorimetric assays are not linked to differences in experimental conditions (i.e. Mn2+ concentration) but are inherent to the methods themselves. However, the overall agreement between the two methods is reasonable.
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5. Conclusion The availability of an efficient and continuous kinetic assay that is able to monitor the AGM-catalyzed reaction will enable enhanced research into enzymes with AGM activity in particular and ureahydrolases in general. The ITC-based assay developed here is practical as it is a rapid and efficient alternative to the colorimetric assay. It is also based on an automated system of timed injection and measurement. Thus, experimental errors introduced into the measurement by a laborintensive method is minimized. Funding This research was supported by the Australian Research Council (DP150104358). DG also acknowledges the Comisión Nacional de Investigación Científica y Tecnológica – CONICYT, Chile, for their support in form of a BECAS postdoctoral fellowship. CRediT authorship contribution statement Liam A. Wilson: Investigation, Writing - original draft. David Garcia: Methodology, Writing - review & editing. Marcelo Monteiro Pedroso: Writing - review & editing, Methodology. Benjamin L. Schulz: Conceptualization, Project administration, Supervision. Luke W. Guddat: Project administration, Supervision. Gerhard Schenk: Project administration, Writing - review & editing, Supervision. References [1] D.E. Wilcox, Binuclear metallohydrolases, Chem. Rev. 96 (1996) 2435–2458, https://doi.org/10.1021/cr950043b.
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