pH-Dependent Thermal Stability of Vibrio cholerae L-asparaginase

Author(s): Remya Radha, Sathyanarayana N. Gummadi*

Journal Name: Protein & Peptide Letters

Volume 26 , Issue 10 , 2019


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Graphical Abstract:


Abstract:

Background: pH is one of the decisive macromolecular properties of proteins that significantly affects enzyme structure, stability and reaction rate. Change in pH may protonate or deprotonate the side group of aminoacid residues in the protein, thereby resulting in changes in chemical and structural features. Hence studies on the kinetics of enzyme deactivation by pH are important for assessing the bio-functionality of industrial enzymes. L-asparaginase is one such important enzyme that has potent applications in cancer therapy and food industry.

Objective: The objective of the study is to understand and analyze the influence of pH on deactivation and stability of Vibrio cholerae L-asparaginase.

Methods: Kinetic studies were conducted to analyze the effect of pH on stability and deactivation of Vibrio cholerae L-asparaginase. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) studies have been carried out to understand the pH-dependent conformational changes in the secondary structure of V. cholerae L-asparaginase.

Results: The enzyme was found to be least stable at extreme acidic conditions (pH< 4.5) and exhibited a gradual increase in melting temperature from 40 to 81 °C within pH range of 4.0 to 7.0. Thermodynamic properties of protein were estimated and at pH 7.0 the protein exhibited ΔG37of 26.31 kcal mole-1, ΔH of 204.27 kcal mole-1 and ΔS of 574.06 cal mole-1 K-1.

Conclusion: The stability and thermodynamic analysis revealed that V. cholerae L-asparaginase was highly stable over a wide range of pH, with the highest stability in the pH range of 5.0–7.0.

Keywords: L-asparaginase, deactivation kinetics, pH stability, half-life time, specific activity, Tm.

[1]
Dubey, V.K.; Jagannadham, M.V. Differences in the unfolding of procerain induced by pH, guanidine hydrochloride, urea, and temperature. Biochemistry, 2003, 42(42), 12287-12297.
[http://dx.doi.org/10.1021/bi035047m] [PMID: 14567690]
[2]
Privalov, P.L. Stability of proteins: Small globular proteins. Adv. Protein Chem., 1979, 33, 167-241.
[http://dx.doi.org/10.1016/S0065-3233(08)60460-X] [PMID: 44431]
[3]
Boer, H.; Koivula, A. The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A. Eur. J. Biochem., 2003, 270(5), 841-848.
[http://dx.doi.org/10.1046/j.1432-1033.2003.03431.x] [PMID: 12603317]
[4]
Talley, K.; Alexov, E. On the pH-optimum of activity and stability of proteins. Proteins, 2010, 78(12), 2699-2706.
[http://dx.doi.org/10.1002/prot.22786] [PMID: 20589630]
[5]
Sadana, A. Biocatalysis: Fundamentals of Enzyme Deactvation Kinetics; Prentice-Hall, Inc: Englewood Cliffs, New Jersey, 1995.
[6]
Gummadi, S.N. What is the role of thermodynamics on protein stability? Biotechnol. Bioprocess Eng., 2003, 8, 9-18.
[http://dx.doi.org/10.1007/BF02932892]
[7]
Teles, R.C.L. Calderon, Lde.A.; Medrano, F.J.; Barbosa, J.A.; Guimarães, B.G.; Santoro, M.M.; de Freitas, S.M. pH dependence thermal stability of a chymotrypsin inhibitor from Schizolobium parahyba seeds. Biophys. J., 2005, 88(5), 3509-3517.
[http://dx.doi.org/10.1529/biophysj.104.045682] [PMID: 15764660]
[8]
Onishi, Y.; Prihanto, A.A.; Yano, S.; Takagi, K.; Umekawa, M.; Wakayama, M. Effective treatment for suppression of acrylamide formation in fried potato chips using L-asparaginase from Bacillus subtilis. 3 Biotech., 5, 783-789.2015
[9]
Egler, R.A.; Ahuja, S.P.; Matloub, Y. L-asparaginase in the treatment of patients with acute lymphoblastic leukemia. J. Pharmacol. Pharmacother., 2016, 7(2), 62-71.
[http://dx.doi.org/10.4103/0976-500X.184769] [PMID: 27440950]
[10]
Pieters, R.; Hunger, S.P.; Boos, J.; Rizzari, C.; Silverman, L.; Baruchel, A.; Goekbuget, N.; Schrappe, M.; Pui, C-H. L-asparaginase treatment in acute lymphoblastic leukemia: A focus on Erwinia asparaginase. Cancer, 2011, 117(2), 238-249.
[http://dx.doi.org/10.1002/cncr.25489] [PMID: 20824725]
[11]
Wang, H.; Li, D.; Li, J-T.; Wang, X-L.; Hao, L-C. Side effects of L-asparaginase during therapies for remission induction and maintenance in children with acute lymphocytic leukemia. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2009, 17(3), 739-741.
[PMID: 19549398]
[12]
Ko, R.H.; Jones, T.L.; Radvinsky, D.; Robison, N.; Gaynon, P.S.; Panosyan, E.H.; Avramis, I.A.; Avramis, V.I.; Rubin, J.; Ettinger, L.J.; Seibel, N.L.; Dhall, G. Allergic reactions and antiasparaginase antibodies in children with high-risk acute lymphoblastic leukemia: A children’s oncology group report. Cancer, 2015, 121(23), 4205-4211.
[http://dx.doi.org/10.1002/cncr.29641] [PMID: 26308766]
[13]
Ghasemi, A.; Asad, S.; Kabiri, M.; Dabirmanesh, B. Cloning and characterization of Halomonas elongata L-asparaginase, a promising chemotherapeutic agent. Appl. Microbiol. Biotechnol., 2017, 101(19), 7227-7238.
[http://dx.doi.org/10.1007/s00253-017-8456-5] [PMID: 28801829]
[14]
Radha, R.; Arumugam, N.; Gummadi, S.N. Glutaminase free L-asparaginase from Vibrio cholerae: Heterologous expression, purification and biochemical characterization. Int. J. Biol. Macromol., 2018, 111, 129-138.
[http://dx.doi.org/10.1016/j.ijbiomac.2017.12.165] [PMID: 29307802]
[15]
Nagarajan, A.; Thirunavuk, N.; Suryanarayanan, T.S.; Gummadi, S.N. Screening and isolation of novel glutaminase free L-asparaginase from fungal endophytes. Res. J. Microbiol., 2014, 9, 163-176.
[http://dx.doi.org/10.3923/jm.2014.163.176]
[16]
Louis-Jeune, C.; Andrade-Navarro, M.A.; Perez-Iratxeta, C. Prediction of protein secondary structure from circular dichroism using theoretically derived spectra. Proteins, 2012, 80(2), 374-381.
[http://dx.doi.org/10.1002/prot.23188] [PMID: 22095872]
[17]
Huynh, K.; Partch, C.L. Analysis of protein stability and ligand interactions by thermal shift assay. Curr. Protoc. Protein Sci., 79, 1-14.2015,
[18]
Gill, P.; Moghadam, T.T.; Ranjbar, B. Differential scanning calorimetry techniques: applications in biology and nanoscience. J. Biomol. Tech., 2010, 21(4), 167-193.
[PMID: 21119929]
[19]
Kotzia, G.A.; Labrou, N.E. Cloning, expression and characterisation of Erwinia carotovora L-asparaginase. J. Biotechnol., 2005, 119(4), 309-323.
[http://dx.doi.org/10.1016/j.jbiotec.2005.04.016] [PMID: 15951039]
[20]
Li, Y.H.; Yu, C.Y.; Li, X.X.; Zhang, P.; Tang, J.; Yang, Q.; Fu, T.; Zhang, X.; Cui, X.; Tu, G.; Zhang, Y.; Li, S.; Yang, F.; Sun, Q.; Qin, C.; Zeng, X.; Chen, Z.; Chen, Y.Z.; Zhu, F. Therapeutic target database update 2018: enriched resource for facilitating bench-to-clinic research of targeted therapeutics. Nucleic Acids Res., 2018, 46(D1), D1121-D1127.
[PMID: 29140520]
[21]
Gwinn, D.M.; Lee, A.G.; Briones-Martin-Del-Campo, M.; Conn, C.S.; Simpson, D.R.; Scott, A.I.; Le, A.; Cowan, T.M.; Ruggero, D.; Sweet-Cordero, E.A. Oncogenic KRAS regulates amino acid homeostasis and asparagine biosynthesis via ATF4 and alters sensitivity to L-asparaginase. Cancer Cell, 2018, 33(1), 91-107.
[http://dx.doi.org/10.1016/j.ccell.2017.12.003] [PMID: 29316436]
[22]
Kishore, D.; Kundu, S.; Kayastha, A.M. Thermal, chemical and pH induced denaturation of a multimeric β-galactosidase reveals multiple unfolding pathways. PLoS One, 2012, 7(11)e50380
[http://dx.doi.org/10.1371/journal.pone.0050380] [PMID: 23185611]
[23]
Manning, M.C.; Patel, K.; Borchardt, R.T. Stability of protein pharmaceuticals. Pharm. Res., 1989, 6(11), 903-918.
[http://dx.doi.org/10.1023/A:1015929109894] [PMID: 2687836]
[24]
Vinothkumar, K.R.; Henderson, R. Structures of membrane proteins. Q. Rev. Biophys., 2010, 43(1), 65-158.
[http://dx.doi.org/10.1017/S0033583510000041] [PMID: 20667175]
[25]
Barrera, F.N.; Weerakkody, D.; Anderson, M.; Andreev, O.A.; Reshetnyak, Y.K.; Engelman, D.M. Roles of carboxyl groups in the transmembrane insertion of peptides. J. Mol. Biol., 2011, 413(2), 359-371.
[http://dx.doi.org/10.1016/j.jmb.2011.08.010] [PMID: 21888917]
[26]
Barroca, M.; Santos, G.; Johansson, B.; Gillotin, F.; Feller, G.; Collins, T. Deciphering the factors defining the pH-dependence of a commercial glycoside hydrolase family 8 enzyme. Enzyme Microb. Technol., 2017, 96, 163-169.
[http://dx.doi.org/10.1016/j.enzmictec.2016.10.011] [PMID: 27871378]
[27]
Kumar, D.P.; Tiwari, A.; Bhat, R. Effect of pH on the stability and structure of yeast hexokinase A. Acidic amino acid residues in the cleft region are critical for the opening and the closing of the structure. J. Biol. Chem., 2004, 279(31), 32093-32099.
[http://dx.doi.org/10.1074/jbc.M313449200] [PMID: 15145950]


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Article Details

VOLUME: 26
ISSUE: 10
Year: 2019
Published on: 29 September, 2019
Page: [743 - 750]
Pages: 8
DOI: 10.2174/0929866526666190617092944
Price: $65

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