Enzyme Kinetics of Recombinant Dihydroorotase from Methanococcus jannaschii


  • Seth A. Ayotte Department of Physics and Department of Chemical Engineering, Cleveland State University
  • Jacqueline Vitali Department of Physics and Department of Biological, Geological and Environmental Sciences, Cleveland State University




Archaea, Methanococcus jannaschii, Dihydroorotase, Pyrimidine metabolism


Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl-L aspartate (CA) to L-dihydroorotate (DHO) in the third step of de novo pyrimidine biosynthesis. The reaction is pH dependent; at low pH the biosynthetic reaction is favored (CA to DHO) and at high pH the degradative reaction is favored (DHO to CA). Even though DHOases share a common catalytic mechanism, they form a very diverse family of proteins. Methanococcus jannaschii is a hyperthermophilic and barophilic archaeon and its DHOase (Mj DHOase) is the first archaeal one that is being studied. A previously conducted physicochemical characterization of Mj DHOase gave information into its similarities and differences from the other known DHOases. In that study, enzyme kinetics were only analyzed in the degradative direction. The purpose of the current work is to further characterize Mj DHOase by studying the kinetics in the biosynthetic direction and the dependence of the reaction on pH in both directions. The properties obtained are compared with other known DHOases. The specific activity of Mj DHOase in the biosynthetic direction is approximately half the specific activity in the degradative direction, similar to human DHOase. Mj DHOase exhibits the characteristic pH dependence of the reaction.