Evidence supporting lysine 166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase as the essential base which initiates catalysis.
The epsilon-amino group of Lys-166 of Rhodospirillum 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-arabinitol 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.
D001665:Binding Sites; D005998:Glycine; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D009154:Mutation; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*
D046911:Macromolecular Substances; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine; D005998:Glycine