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    • Keywords


      203-214Ribulose-P2 carboxylase; site-directed mutagenesis; essentiality of Lys-166; non-essentiality of His-291

    • Abstract


      Both Lys-166 and His-291 of ribulosebisphosphate carboxylase/oxygenase fromRhodospirillum rubrum have been implicated as the active-site residue that initiates catalysis. To decide between these two candidates, we resorted to site-directed mutagenesis to replace Lys-166 and His-291 with several amino acids. All 7 of the position-166 mutants tested are severely deficient in carboxylase activity, whereas the alanine and serine mutants at position 291 are ∼40% and ∼18% as active as the native carboxylase, essentially ruling out His-291 in theRhodospirillum rubrum carboxylase (and by inference His-298 in the spinach enzyme) as a catalytically essential residue. The ability of some of the mutant proteins to undergo carbamate formation or to bind either ribulosebisphosphate or a transition-state analogue remains largely unimpaired. This implies that Lys-166 is not required for substrate binding; rather, the results corroborate the earlier postulate that Lys-166 functions as an acid-base group in catalysis or in stabilizing a transition state in the reaction pathway.

    • Author Affiliations


      Salil K Niyogi1 Thomas S Soper1 Robert S Foote1 Frank W Larimer1 Richard J Mural1 Sankar Mitra1 Eva H Lee1 Richard Machanoff1 Fred C Hartman1

      1. The Protein Engineering and Molecular Mutagenesis Program of the Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee - 37831, USA
    • Dates

  • Journal of Biosciences | News

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