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Evidence supporting lysine 166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase as the essential base which initiates catalysis


, : Evidence supporting lysine 166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase as the essential base which initiates catalysis. Journal of Biological Chemistry 263(14): 6468-6471

The .epsilon.-amino group of Lys-166 of Rhosdospirillum rubrum ribulosebisphosphate carboxylase/oxygenase was postulated as the essential base which initiates catalysis by abstracting the proton at C-3 of ribulose 1,5-bisphosphate (Hartman, F. C., Soper, T. S., Niyogi, S. K., Mural, R. J., Foote, R.S., Mitra, S., Lee, E. H., Machanoff, R., and Larimer, F. W. (1987) J. Biol Chem. 262, 3496-3501). To scrutinize this possibility, the site-directed Gly-166 mutant, totally devoid of ribulosebisphosphate carboxylase activity, was examined for its ability to catalyze each of three partial reactions. When carbamylated at Lys-191 (i.e. activated with CO2 and Mg2+), wild-type enzyme catalyzed the hydrolysis of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate, the six-carbon reaction intermediate of the carboxylase reaction (Pierce, J., Andrews, T. J., and Lorimer, G. H. (1986a) J. Biol. Chem. 261, 10248-10256). Likewise, when carbamylated at Lys-191, the Gly-166 mutant also catalyzed the hydrolysis of this reaction intermediate. The carbamylated wild type catalyzed the enolization of ribulose 1,5-bisphosphate as indicated by the transfer of 3H radioactivity from [3-3H]ribulose, 1,5-bisphosphate to the medium. However, even when carbamylated at Lys-191, the mutant protein did not catalyze the enolization of ribulose 1,5-bisphosphate. Additionally, unlike the decarbamylated wild-type enzyme, which catalyzed the decarboxylation of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate in the absence of Mg2+, the mutant protein was inactive in this partial reaction. These properties exclude the .epsilon.-amino group of Lys-166 as an obligatory participant in the hydrolysis of 2-carboxy-3-keto-D-arabintol 1,5-bisphosphate. In contrast, these properties are consistent with the .epsilon.-amino group of Lys-166 functioning as an acid-base catalyst in the enolization of ribulose 1,5-bisphosphate(when the enzyme is carbamylated) and in the decarboxylation of 2-carboxy-3-keto-D-arabinitol 1,5-bisphosphate (when the enzyme is decarbamylated). Alternatively Lys-166 may stabilize the transition states of these two partial reactions.

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Accession: 005411931

PMID: 3129424

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Related references

Hartman F.C.; Mural R.J.; Mitra S.; Soper T.S.; Niyogi S.K.; Foote R.S.; Machanoff R.; Lee E.H.; Larimer F.W., 1986: Site directed mutagenesis of lysine 166 of ribulosebisphosphate carboxylase oxygenase from rhodospirillum rubrum. Federation Proceedings 45(6): 1913

Schloss, J.V.; Phares, E.F.; Long, M.V.; Norton, I.L.; Stringer, C.D.; Hartman, F.C., 1982: Ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. Methods in Enzymology 90 Pt E: 522-528

Morell, M.K.; Kane, H.J.; Andrews, T.J., 1990: Carboxylterminal deletion mutants of ribulosebisphosphate carboxylase from Rhodospirillum rubrum. The carboxylterminal octapeptide of ribulosebisphosphate carboxylase from Rhodospirillum rubrum, which lacks small subunits, shows homology to a highly conserved region near the amino terminus of the small subunits of hexadecameric ribulosebisphos...

Hartman, F.C.; Stringer, C.D.; Lee, E.H., 1984: Complete primary structure of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. Of the 14 cyanogen bromide fragments derived from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase, four are too large to permit complete sequencing by direct means [F. C. Hartman, C. D. Stringer, J. Omnaas, M. I. Donnelly, and B....

Day, A.G.; Chène, P.; Fersht, A.R., 1993: Role of phenylalanine-327 in the closure of loop 6 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. Phenylalanine-327 of ribulosebisphosphate carboxylase/oxygenase (rubisco) from Rhodospirillum rubrum was mutated to tryptophan, leucine, valine, alanine, and glycine, and was also deleted. The least active mutant, the deletion mutant, exhibits les...

Schloss, J.V.; Phares, E.F.; Long, M.V.; Norton, I.L.; Stringer, C.D.; Hartman, F.C., 1979: Isolation, characterization, and crystallization of ribulosebisphosphate carboxylase from autotrophically grown Rhodospirillum rubrum. Serial culture of Rhodospirillum rubrum with 2% CO2 in H2 as the exclusive carbon source resulted in a rather large fraction of the soluble protein (greater than 40%) being comprised of ribulosebisphosphate carboxylase (about sixfold higher than t...

O'leary, M.H.; Jaworski, R.J.; Hartman, F.C., 1979: C nuclear magnetic resonance study of the CO(2) activation of ribulosebisphosphate carboxylase from Rhodospirillum rubrum. Ribulosebisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from Rhodospirillum rubrum is activated by CO(2) and Mg(2+). (13)C NMR spectra were determined for the unactivated enzyme and for enzyme that had been...

Day, A.G.; Chene, P.; Fersht, A.R., 1993: Role of phenylalanine-327 in the closure of loop 6 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. Phenylalanine-327 of ribulosebisphosphate carboxylase/oxygenase (rubisco) from Rhodospirillum rubrum was mutated to tryptophan, leucine, valine, alanine, and glycine, and was also deleted. The least active mutant, the deletion mutant, exhibits les...

Niyogi S.K.; Foote R.S.; Mural R.J.; Larimer F.W.; Mitra S.; Soper T.S.; Machanoff R.; Hartman F.C., 1986: Site directed mutagenesis of histidine 291 of ribulosebisphosphate carboxylase oxygenase from rhodospirillum rubrum. Federation Proceedings 45(6): 1913

Herndon, C.S.; Norton, I.L.; Hartman, F.C., 1982: Reexamination of the binding site for pyridoxal 5'-phosphate in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. The high specificity of pyridoxal 5'-phosphate (PLP) for an essential lysyl residue of ribulosebisphosphate carboxylase/oxygenase was confirmed, but half-of-sites reactivity was not observed in contrast to an earlier report [Robison, P. D., W...