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Purification and biochemical properties of an N-hydroxyarylamine O-acetyltransferase from Escherichia coli |
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| Authors: | Ei-Tora Yamamura Michio Sayama Makiko Kakikawa Masa-aki Mori Akira Taketo Ken-Ichi Kodaira |
| Affiliation: | 1. Molecular Biology Group, Faculty of Engineering, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan;2. School of Health Sciences, Kyusyu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan;3. Department of Biochemistry 1, Fukui Medical University, Matsuoka-cho, Yoshida-gun, Fukui 910-1193, Japan |
| Abstract: | The N-hydroxyarylamine O-acetyltransferase of Escherichia coli has been expressed as a histidine tagged fusion protein and purified using immobilized nickel column chromatography. The molecular mass of the histidine tagged N-hydroxyarylamine O-acetyltransferase was estimated to be 60.0 kDa by gel filtration and 34.0 kDa by SDS–PAGE and DNA sequence, suggesting that the native enzyme exists as homo dimer. The catalytic properties were investigated using o-aminobenzoic acid as a substrate. No difference in acetyltransfer activity was observed between histidine tagged protein and untagged enzyme. Kinetic studies indicated a ping-pong bi bi mechanism of the catalysis. Inhibition by N-ethylmaleimide and salicylic acid was competitive with o-aminobenzoic acid and non-competitive with acetyl-CoA. |
| Keywords: | Ping-pong bi bi Histidine tag Dimer Acetyl-CoA |
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