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KMID : 0380219930260020176
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Journal of Biochemistry and Molecular Biology
1993 Volume.26 No. 2 p.176 ~ p.183
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Malonyl-CoA Synthetase from Rhizobium trifolii: Purificatin, Properties, and the Immunological Comparison with Those from Bradyrhizobium japonicum and Pseudomonas fluorescens
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Yu Sam Kim
Sun Jong Kwon and Sang Won Kang
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Abstract
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Malonyl-CoA synthetase which catalyzes the formation of malonyl-CoA, AMP, and Ppi directly from malonate and CoA in the presence of ATP was purified from a symbiotic bacteria with clover, rhizobium trifolii, grown on glucose as a carbon source, The enzyme in the buffer containing 15% glycerol was stable for 6 months when it was kept in -20。C freezer. The molecular size of the enzyme was 63,000 Da which was monomeric. N-terminal sequence of the enzyme was SNHLFDAMRAAAPGN-, Alanine content (13.8mol%) in the enzyme was the highest. The molar extinction coefficient at 280 nm for native enzyme was 5.03×10E4M-1.cm-1. This enzyme was highly specific on malonate, CoAn and ATP as substrate. The optimum pH was 8.4. Kinetic constants, Km, for malonate, coa, an ATP were 216.9 M, 13.8 M, and 31.4 M, respectively. It required magnesium or manganese ion for its catalysis. Diethylpyrocarbonate and pyridoxal-5'-phosphate inactivated the enzyme activity, indicating that imidazole and amine groups are estimated to be essential. It was immunogenic to rabbit and the antibody and amine groups are estimated to be essential. It was immunogenic to rabbit and the antibody raised with this enzyme inactivated and precipitate malonyl-CoA synthetase from Bradyrhizobium japonicum and Pseudomonds fluorescens. It suggests that there are some immunological identity among malonyl-CoA synthtases from three different bacteria.
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KEYWORD
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