Purification and Characterization of Bacillus subtilis Protoporphyrinogen Oxidase and Pre-equilibrium Behavior During Oxidation of Protoporphyrinogen IX

  • Jeong, Eun-Ju (Department of Genetic Engineering and Biotechnology Research Institute, Chonnarn National University) ;
  • Han, Ok-Soo (Department of Genetic Engineering and Biotechnology Research Institute, Chonnarn National University)
  • 투고 : 2000.10.30
  • 심사 : 2000.11.28
  • 발행 : 2001.01.31

초록

Previous studies indicate that B. subtilis protoporphyrinogen oxidase is poorly inhibited by diphenyl ether herbicides. To better understand the basis of this insensitivity, the enzyme was overexpressed as a soluble protein in E. coli, purified and characterized. The mechanism of oxidation of B. subtilis protoporphyrinogen IX was studied and the enzyme kinetic parameters were determined for protoporpyrinogen IX; $K_m$, and $k_{cat}$ were $6.3\;{\mu}M$ and $0.028\;h-^1$, respectively. The enzyme required flavin adenine dinucleotide as a cofactor and its activity was enhanced by 1 mM n-octylglucopyranoside. The nonenzymatic oxidation rate was dependent on the concentration of protoporphyrinogen IX, suggesting that the reaction involves a pre-equilibrium step followed by a rate-limiting step.

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