Evidence supporting lysine 166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase as the essential base which initiates catalysis
The element of-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 or ribulose 1,5-bisphosphate. 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 CO/sub 2/ and Mg/sup 2 +/), 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. 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 /sup 3/H radioactivity from (3-/sup 3/H)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. These properties exclude the element of-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 element of-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.
- Research Organization:
- E. I. du Pont de Nemours and Co., Wilmington, DE (USA)
- DOE Contract Number:
- AC05-84OR21400
- OSTI ID:
- 6925224
- Journal Information:
- J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 263:14; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ALCOHOLS
AMINO ACIDS
BACTERIA
BIOCHEMICAL REACTION KINETICS
CARBON-CARBON LYASES
CARBOXY-LYASES
CARBOXYLIC ACIDS
CATALYSIS
ENOLS
ENZYMES
HYDROXY COMPOUNDS
KINETICS
LABELLED COMPOUNDS
LYASES
LYSINE
MICROORGANISMS
MOLECULAR STRUCTURE
MUTANTS
ORGANIC ACIDS
ORGANIC COMPOUNDS
REACTION KINETICS
RHODOSPIRILLUM
RIBULOSE DIPHOSPHATE CARBOXYLASE
TRITIUM COMPOUNDS