Soybean (Glycine max) urease: Significance of sulfhydryl groups in urea catalysis |
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| Authors: | Sandeep Kumar Arvind M Kayastha |
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| Institution: | 1. H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan;2. Institute of Chemical Sciences, Bahauddin Zakariya University, Multan, Pakistan;3. Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan;4. Department of Life Science Informatics, B-IT, LIMES Program Unit Chemical Biology and Medicinal Chemistry, Rheinische Friedrich-Wilhelms-Universität, Dahlmannstr. 2, D-53113 Bonn, Germany;5. Pharmaceutical Institute, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany;6. Department of Physics, University of Sargodha, Sargodha, Pakistan;1. Programa de Pós-Graduação em Genética e Biologia Molecular, Departamento de Genética, Instituto de Biociências, Universidade Federal do Rio Grande do Sul (UFRGS), 91501-970, Porto Alegre, RS, Brazil;2. Programa de Pós-Graduação em Biologia Celular e Molecular, Centro de Biotecnologia, UFRGS, 91501-970, Porto Alegre, RS, Brazil;3. Instituto do Cérebro, Pontifícia Universidade do Rio Grande do Sul (PUCRS), Av. Ipiranga 6690, 90610-000, Porto Alegre, Brazil;1. Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan;2. National Center for Bioinformatics, Quaid-i-Azam University, Islamabad 45320, Pakistan;3. Department of Chemistry, Gomal University, Dera Ismail Khan 29050, Pakistan;4. Department of Physics, Sargodha University, Sargodha 40100, Pakistan;1. Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia;2. Department of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul Wali Khan University Mardan, Pakistan;1. Department of Chemistry, Hazara University, Mansehra 21120, Khyber Pukhtankhwa, Pakistan;2. Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad 22060, KPK, Pakistan;3. Department of Biochemistry and Biotechnology, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan;4. Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), PuncakAlam Campus, 42300 Bandar PuncakAlam, Selangor DarulEhsan, Malaysia;5. Faculty of Applied Science, UiTM, 40450 Shah Alam, Selangor, Malaysia;6. Department of Environmental Science, COMSATS Institute of Information Technology, Abbottabad 22060, KPK, Pakistan |
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| Abstract: | The soybean urease (urea amidohydrolase; EC 3.5.1.5) was investigated to elucidate the presence of sulfhydryl (–SH) groups and their significance in urea catalysis with the help of various –SH group specific reagents. The native urease incubated with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) showed exponential increase in the absorbance, thereby revealing the presence of –SH groups. A total of 34 –SH groups per hexamer enzyme molecule were estimated from the absorption studies which represents nearly six –SH groups per subunit. The time-dependent inactivation of urease with DTNB, p-chloromercuribenzoate (p-CMB), N-ethylmaleimide (NEM) and iodoacetamide (IAM) showed biphasic kinetics, where half of the enzyme activity was lost more rapidly than the other half. This study reveals the presence of two categories of “accessible” –SH groups, one category being more reactive than the other. The inactivation of urease by p-CMB was largely reversed on treatment with cysteine, which might be due to unblocking of –SH group by mercaptide exchange reaction. Finally, when NEM inactivated urease was incubated with sodium fluoride, a time-dependent regain of activity was observed with higher concentrations of fluoride ion. |
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