Thermostable succinyl-Coenzyme A synthetase from Nitrosomonas europaea ATCC 25978: Purification and properties |
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| Institution: | 1. Department of Medical Biochemistry, Semmelweis University, Budapest 1094, Hungary;2. MTA-SE Lendület Hereditary Endocrine Tumours Research Group, Budapest 1088, Hungary;3. MTA-SE Lendület Neurobiochemistry Research Group, Budapest 1094, Hungary;1. Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary;2. 1st Department of Pediatrics, “Hippokratio” General Hospital, Aristotle University, Thessaloniki, Greece;3. Department of Paediatric Metabolic Medicine, Great Ormond Street Hospital, London, UK;4. Department of Pediatrics, Radboud University Medical Centre, Nijmegen, The Netherlands;5. Translational Metabolic Laboratory, Department Laboratory Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands;6. Laboratory Genetic Metabolic Diseases, Amsterdam University Medical Centers, University of Amsterdam, The Netherlands;7. MTA-ELTE Laboratory of Molecular and Systems Neurobiology, Department of Physiology and Neurobiology, Hungarian Academy of Sciences, Eotvos Lorand University, Budapest, Hungary;8. Department of Anatomy, Histology and Embryology, Semmelweis University, Budapest, Hungary |
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| Abstract: | The ammonia-oxidizing chemoautotrophic bacterium Nitrosomonas europaea possesses prominant succinate-reducing activity of succinyl-Coenzyme A synthetase (SCS, EC 6.2.1.5). SCS was purified as an electrophoretically homogeneous protein from Nitrosomonas europaea strain ATCC 25978 about 275-fold, with a 3.9% activity yield. The molecular mass of the native enzyme was estimated to be about 130 kDa by gel filtration, whereas SDS-PAGE gave two protein bands with Mr values of 29 (α) and 36 kDa (β). The isoelectric point of the enzyme was 5.3. The apparent Km values of the enzyme for ATP, succinate and CoA were 0.4 mM, 5 mM and 0.1 mM, respectively. The pH and temperature optima of the SCS were about 5.0 and 55°C, respectively. The SCS was stable in the pH range of 8.0–10.0 and up to 70°C. The enzyme was thermostable; 50% of the enzyme activity was retained at 90–100°C for 10 min. The SCS was activated by Mg2+ at 1.0–100 mM, but inhibited by Cu2+ (0.1 mM) and SDS (1.0 mM). The enzyme utilized ATP as the preferred substrate. |
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