Mol. Cells 2020; 43(9): 784-792
Published online August 31, 2020
https://doi.org/10.14348/molcells.2020.0136
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *nationface@jbnu.ac.kr (YJK); junghee.park@jbnu.ac.kr (JHP)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Arginine kinase (AK), a bioenergy-related enzyme, is distributed widely in invertebrates. The role of highly conserved histidines in AKs is still unascertained. In this study, the highly conserved histidine 284 (H284) in AK of Daphnia magna (DmAK) was replaced with alanine to elucidate the role of H284. We examined the alteration of catalytic activity and structural changes of H284A in DmAK. The catalytic activity of H284A was reduced dramatically compared to that in wild type (WT). Thus the crystal structure of H284A displayed several structural changes, including the alteration of D324, a hydrogen-bonding network around H284, and the disruption of π-stacking between the imidazole group of the H284 residue and the adenine ring of ATP. These findings suggest that such alterations might affect a conformational change of the specific loop consisting of G310-V322 at the antiparallel β-sheet region. Thus, we speculated that the H284 residue might play an important role in the conformational change of the specific loop when ATP binds to the substrate-binding site of DmAK.
Keywords arginine kinase, crystallization, kinetics, point mutation, X-ray crystallography
Phosphagen kinases (PKs) are a family of phosphotransferases that reversibly catalyze the delivery of high-energy phosphoryl groups between ATP and guanidine derivatives. The phosphorylated guanidines, also known as phosphagens, are a type of high-energy compounds that play an important role in cellular energy homeostasis in both the muscles and the brain by providing a way to store high-energy phosphates as a metastable compound (Ellington, 2001). PKs are widespread in all types of animal cells that require considerable amounts of energy. Eight PKs have been reported in invertebrates (Ellington, 1989; Uda et al., 2005). One of these, arginine kinase (AK), is distributed widely in invertebrates, and its activity has also been observed in arthropods, mollusks, nematodes, protozoans, and some bacteria (Ellington, 1989; Newsholme et al., 1978).
Arginine, which is an essential amino acid in many organisms, is produced as an intermediate in two regulatory cycles: the urea and the nitric oxide cycles (Noh et al., 2002). This intermediate is also used by AK to maintain energy homeostasis in cells. AK requires both arginine and ATP as bi-substrates and plays a key role in catalyzing the reversible transfer of the γ-phosphoryl group of ATP to the guanidine functional group of arginine (Adeyemi and Whiteley, 2014; Alonso et al., 2001; Hansen and Knowles, 1981; Wyss and Kaddurah-Daouk, 2000; Zhou et al., 1998). In particular, AK activity is required for muscle contraction and the generation of electrical conductance in nerves. Many functional and structural studies of AKs from diverse organisms have been reported in a substrate-binding-dependent manner (Azzi et al., 2004; Brown and Grossman, 2004; Fernandez et al., 2007; Niu et al., 2011; Pruett et al., 2003; Watts et al., 1980). The overall topology of AKs commonly has two different domains: an N-terminal domain, which consists of small α-helices, and a C-terminal domain, which comprises an eight-strand antiparallel β-sheet connected by α-helices. In AKs, the N-terminal domain can interact with arginine (i.e., either L-arginine or phosphoarginine), whereas the central role of the C-terminal domain is nucleotide-binding (i.e., either ATP or ADP) (Fernandez et al., 2007; Zhou et al., 1998). In the structural comparison between
Several highly conserved residues in the arginine and ATP binding sites that alter AK enzyme activity have been reported (Guo et al., 2004; Strong and Ellington, 1996; Takeuchi et al., 2004; Wu et al., 2014). In
Although several mutagenesis studies have reported highly conserved residues at the arginine binding site of AKs, a role has not yet been elucidated for the highly conserved histidine residue H284 around the ATP binding site. This study is the first report of the crystal structure of wild-type
The
To ascertain the optimal pH and temperature, AK activity was conducted as described previously (Li et al., 2006; Pereira et al., 2000; Wu et al., 2014). For the optimal pH and temperature determination, enzyme activity was measured in 100 mM sodium citrate-citrate buffers at pH 5.5-6.5, 100 mM Tris-HCl buffers at pH 7.0-9.0, and 100 mM glycine-NaOH buffers at pH 9.5-10.0 in the temperature range from the 0°C to 70°C, respectively. The reaction mixture for the AK assay contained freshly prepared 10 mM L-arginine, 2 mM ATP, and 3 mM magnesium acetate in 100 mM Tris-HCl, pH 8.5. To initiate the enzyme reaction, we added 30 μl of
Purified
Data collection for the
The
To date, the roles of highly conserved amino acids, i.e., D62, R193, E225, C271, P272, T273, and E314, in AKs have been elucidated using mutagenesis in previously biochemical and biophysical studies (Guo et al., 2004; Liu et al., 2011; Pruett et al., 2003; Strong and Ellington, 1996; Suzuki et al., 2000a; 2000b; Wu et al., 2008; 2014). Furthermore, five histidine residues are well conserved in PKs. The role of each histidine of creatine kinase (CK) in vertebrates has been reported in biochemical and biophysical studies (Chen et al., 1996; Forstner et al., 1997; Muhlebach et al., 1994). Specifically, histidine residues (H96, H105, H190, H233, and H295) from rabbit muscle CK were studied using site-directed mutagenesis, which was replaced with asparagine (Chen et al., 1996). According to the results, H295N located at the active site of rabbit CK showed dramatically reduced enzyme activity compared with the native form; however, the exact roles of H295 still remains a question (Chen et al., 1996). In this study, we focused on H284 of
The overexpressed
Steady-state kinetic measurements of
Moreover, the dissociation constant (
The crystallized
As mentioned above, the crystal structures of
Even though the overall topologies of
Compared with the
To examine the structural differences between the specific loops of the
In conclusion, the rotamer change of D324 and the alteration of the hydrogen bond network could be induced by the disruption of π-stacking. The disruption of π-stacking in the H284A-Arg structure leads to a reduction in
The authors appreciate the staff of Beamlines 7A at the Pohang Accelerator for their technical assistance during the data collection. This research was supported by the “Research Base Construction Fund Support Program” funded by Jeonbuk National University in 2019.
Z.R. and S.Y.K. designed and performed experiment, collected and analyzed data, and wrote manuscript. X.L. and D.S.K. performed the experiment, collected data. Y.J.K. and J.H.P. designed the experiments, edited the manuscript and managed the project.
The authors have no potential conflicts of interest to disclose.
Comparison of the forward reaction parameters of
Vmax (µmolpimin–1mg–1) | Kcat (s–1) | |||||
---|---|---|---|---|---|---|
111.6 ± 3.61 | 18.6 ± 0.60 | 0.90 ± 0.11 | 0.74 ± 0.10 | 0.47 ± 0.07 | 0.87 ± 0.12 | |
11.66 ± 1.23 | 0.19 ± 0.02 | 0.90 ± 0.45 | 0.41 ± 0.21 | 0.30 ± 0.12 | 1.01 ± 0.44 |
Mol. Cells 2020; 43(9): 784-792
Published online September 30, 2020 https://doi.org/10.14348/molcells.2020.0136
Copyright © The Korean Society for Molecular and Cellular Biology.
Zhili Rao1,4 , So Young Kim1,4
, Xiaotong Li1
, Da Som Kim1
, Yong Ju Kim2,3,*
, and Jung Hee Park1,3,*
1Division of Biotechnology, College of Environmental & Bioresources Sciences, Jeonbuk National University, Iksan 54596, Korea, 2Department of Herbal Medicine Resources, College of Environmental and Bioresource Sciences, Jeonbuk National University, Iksan 54596, Korea, 3Advanced Institute of Environment and Bioscience, College of Environmental & Bioresources Sciences, Jeonbuk National University, Iksan 54596, Korea, 4These authors contributed equally to this work.
Correspondence to:*nationface@jbnu.ac.kr (YJK); junghee.park@jbnu.ac.kr (JHP)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Arginine kinase (AK), a bioenergy-related enzyme, is distributed widely in invertebrates. The role of highly conserved histidines in AKs is still unascertained. In this study, the highly conserved histidine 284 (H284) in AK of Daphnia magna (DmAK) was replaced with alanine to elucidate the role of H284. We examined the alteration of catalytic activity and structural changes of H284A in DmAK. The catalytic activity of H284A was reduced dramatically compared to that in wild type (WT). Thus the crystal structure of H284A displayed several structural changes, including the alteration of D324, a hydrogen-bonding network around H284, and the disruption of π-stacking between the imidazole group of the H284 residue and the adenine ring of ATP. These findings suggest that such alterations might affect a conformational change of the specific loop consisting of G310-V322 at the antiparallel β-sheet region. Thus, we speculated that the H284 residue might play an important role in the conformational change of the specific loop when ATP binds to the substrate-binding site of DmAK.
Keywords: arginine kinase, crystallization, kinetics, point mutation, X-ray crystallography
Phosphagen kinases (PKs) are a family of phosphotransferases that reversibly catalyze the delivery of high-energy phosphoryl groups between ATP and guanidine derivatives. The phosphorylated guanidines, also known as phosphagens, are a type of high-energy compounds that play an important role in cellular energy homeostasis in both the muscles and the brain by providing a way to store high-energy phosphates as a metastable compound (Ellington, 2001). PKs are widespread in all types of animal cells that require considerable amounts of energy. Eight PKs have been reported in invertebrates (Ellington, 1989; Uda et al., 2005). One of these, arginine kinase (AK), is distributed widely in invertebrates, and its activity has also been observed in arthropods, mollusks, nematodes, protozoans, and some bacteria (Ellington, 1989; Newsholme et al., 1978).
Arginine, which is an essential amino acid in many organisms, is produced as an intermediate in two regulatory cycles: the urea and the nitric oxide cycles (Noh et al., 2002). This intermediate is also used by AK to maintain energy homeostasis in cells. AK requires both arginine and ATP as bi-substrates and plays a key role in catalyzing the reversible transfer of the γ-phosphoryl group of ATP to the guanidine functional group of arginine (Adeyemi and Whiteley, 2014; Alonso et al., 2001; Hansen and Knowles, 1981; Wyss and Kaddurah-Daouk, 2000; Zhou et al., 1998). In particular, AK activity is required for muscle contraction and the generation of electrical conductance in nerves. Many functional and structural studies of AKs from diverse organisms have been reported in a substrate-binding-dependent manner (Azzi et al., 2004; Brown and Grossman, 2004; Fernandez et al., 2007; Niu et al., 2011; Pruett et al., 2003; Watts et al., 1980). The overall topology of AKs commonly has two different domains: an N-terminal domain, which consists of small α-helices, and a C-terminal domain, which comprises an eight-strand antiparallel β-sheet connected by α-helices. In AKs, the N-terminal domain can interact with arginine (i.e., either L-arginine or phosphoarginine), whereas the central role of the C-terminal domain is nucleotide-binding (i.e., either ATP or ADP) (Fernandez et al., 2007; Zhou et al., 1998). In the structural comparison between
Several highly conserved residues in the arginine and ATP binding sites that alter AK enzyme activity have been reported (Guo et al., 2004; Strong and Ellington, 1996; Takeuchi et al., 2004; Wu et al., 2014). In
Although several mutagenesis studies have reported highly conserved residues at the arginine binding site of AKs, a role has not yet been elucidated for the highly conserved histidine residue H284 around the ATP binding site. This study is the first report of the crystal structure of wild-type
The
To ascertain the optimal pH and temperature, AK activity was conducted as described previously (Li et al., 2006; Pereira et al., 2000; Wu et al., 2014). For the optimal pH and temperature determination, enzyme activity was measured in 100 mM sodium citrate-citrate buffers at pH 5.5-6.5, 100 mM Tris-HCl buffers at pH 7.0-9.0, and 100 mM glycine-NaOH buffers at pH 9.5-10.0 in the temperature range from the 0°C to 70°C, respectively. The reaction mixture for the AK assay contained freshly prepared 10 mM L-arginine, 2 mM ATP, and 3 mM magnesium acetate in 100 mM Tris-HCl, pH 8.5. To initiate the enzyme reaction, we added 30 μl of
Purified
Data collection for the
The
To date, the roles of highly conserved amino acids, i.e., D62, R193, E225, C271, P272, T273, and E314, in AKs have been elucidated using mutagenesis in previously biochemical and biophysical studies (Guo et al., 2004; Liu et al., 2011; Pruett et al., 2003; Strong and Ellington, 1996; Suzuki et al., 2000a; 2000b; Wu et al., 2008; 2014). Furthermore, five histidine residues are well conserved in PKs. The role of each histidine of creatine kinase (CK) in vertebrates has been reported in biochemical and biophysical studies (Chen et al., 1996; Forstner et al., 1997; Muhlebach et al., 1994). Specifically, histidine residues (H96, H105, H190, H233, and H295) from rabbit muscle CK were studied using site-directed mutagenesis, which was replaced with asparagine (Chen et al., 1996). According to the results, H295N located at the active site of rabbit CK showed dramatically reduced enzyme activity compared with the native form; however, the exact roles of H295 still remains a question (Chen et al., 1996). In this study, we focused on H284 of
The overexpressed
Steady-state kinetic measurements of
Moreover, the dissociation constant (
The crystallized
As mentioned above, the crystal structures of
Even though the overall topologies of
Compared with the
To examine the structural differences between the specific loops of the
In conclusion, the rotamer change of D324 and the alteration of the hydrogen bond network could be induced by the disruption of π-stacking. The disruption of π-stacking in the H284A-Arg structure leads to a reduction in
The authors appreciate the staff of Beamlines 7A at the Pohang Accelerator for their technical assistance during the data collection. This research was supported by the “Research Base Construction Fund Support Program” funded by Jeonbuk National University in 2019.
Z.R. and S.Y.K. designed and performed experiment, collected and analyzed data, and wrote manuscript. X.L. and D.S.K. performed the experiment, collected data. Y.J.K. and J.H.P. designed the experiments, edited the manuscript and managed the project.
The authors have no potential conflicts of interest to disclose.
. Comparison of the forward reaction parameters of
Vmax (µmolpimin–1mg–1) | Kcat (s–1) | |||||
---|---|---|---|---|---|---|
111.6 ± 3.61 | 18.6 ± 0.60 | 0.90 ± 0.11 | 0.74 ± 0.10 | 0.47 ± 0.07 | 0.87 ± 0.12 | |
11.66 ± 1.23 | 0.19 ± 0.02 | 0.90 ± 0.45 | 0.41 ± 0.21 | 0.30 ± 0.12 | 1.01 ± 0.44 |
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