Site-directed mutagenesis to determine essential residues of ribulose-bisphosphate carboxylase of Rhodospirillum rubrum

Salil K. Niyogi, Thomas S. Soper, Robert S. Foote, Frank W. Larimer, Richard J. Mural, Sankar Mitra, Eva H. Lee, Richard Machanoff, Fred C. Hartman

Research output: Contribution to journalArticle

Abstract

Both Lys-166 and His-291 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum 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 the Rhodospirillum 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.

Original languageEnglish (US)
Pages (from-to)203-214
Number of pages12
JournalJournal of Biosciences
Volume11
Issue number1-4
DOIs
StatePublished - Mar 1 1987

Keywords

  • 203-214Ribulose-P carboxylase
  • essentiality of Lys-166
  • non-essentiality of His-291
  • site-directed mutagenesis

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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