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.
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Abbreviations
- ss:
-
Single-stranded
- ribulose-P2 carboxylase:
-
D-ribulose 1,5-bisphosphate carboxy-lase/oxygenase
- rbc gene:
-
gene for ribulose-P2 carboxylase
- Td:
-
temperature of dissociation
- carboxy-arabinitol-P2 :
-
2-carboxyarabinitol 1, 5-bisphosphate
- RF:
-
replication form
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Niyogi, S.K., Soper, T.S., Foote, R.S. et al. Site-directed mutagenesis to determine essential residues of ribulose-bisphosphate carboxylase ofRhodospirillum rubrum . J. Biosci. 11, 203–214 (1987). https://doi.org/10.1007/BF02704670
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DOI: https://doi.org/10.1007/BF02704670