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

Abstract

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 themore » enzyme is decarbamylated). Alternatively, Lys-166 may stabilize the transition states of these two partial reactions.« less

Authors:
;
Publication Date:
Research Org.:
E. I. du Pont de Nemours and Co., Wilmington, DE (USA)
OSTI Identifier:
6925224
DOE Contract Number:  
AC05-84OR21400
Resource Type:
Journal Article
Journal Name:
J. Biol. Chem.; (United States)
Additional Journal Information:
Journal Volume: 263:14
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; RHODOSPIRILLUM; MOLECULAR STRUCTURE; MUTANTS; RIBULOSE DIPHOSPHATE CARBOXYLASE; BIOCHEMICAL REACTION KINETICS; CATALYSIS; ENOLS; LYSINE; TRITIUM COMPOUNDS; ALCOHOLS; AMINO ACIDS; BACTERIA; CARBON-CARBON LYASES; CARBOXY-LYASES; CARBOXYLIC ACIDS; ENZYMES; HYDROXY COMPOUNDS; KINETICS; LABELLED COMPOUNDS; LYASES; MICROORGANISMS; ORGANIC ACIDS; ORGANIC COMPOUNDS; REACTION KINETICS; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Lorimer, G H, and Hartman, F C. Evidence supporting lysine 166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase as the essential base which initiates catalysis. United States: N. p., 1988. Web.
Lorimer, G H, & Hartman, F C. Evidence supporting lysine 166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase as the essential base which initiates catalysis. United States.
Lorimer, G H, and Hartman, F C. Sun . "Evidence supporting lysine 166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase as the essential base which initiates catalysis". United States.
@article{osti_6925224,
title = {Evidence supporting lysine 166 of Rhodospirillum rubrum ribulosebisphosphate carboxylase as the essential base which initiates catalysis},
author = {Lorimer, G H and Hartman, F C},
abstractNote = {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.},
doi = {},
journal = {J. Biol. Chem.; (United States)},
number = ,
volume = 263:14,
place = {United States},
year = {1988},
month = {5}
}