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Title: Site-directed mutagenesis of Lysine{sup 382}, the activator-binding site, of ADP-Glucose pyrophosphorylase from Anabaena PCC 6120

Abstract

Previous studies have shown that a highly conserved lysyl residue (Lys{sup 419}) near the C-terminus of Anabaena ADP-glucose pyrophosphorylase is involved in the binding of 3-P-glycerate, the allosteric activator. Phosphopyridoxylation of the K419R mutant enzyme modified another conserved lysyl residue (Lys{sup 382}), suggesting that this residue might be also located within the activator-binding site. Site-directed mutagenesis of Lys{sup 382} of the Anabaena enzyme was performed to determine the role of this residue. Replacing Lys{sup 382} with either arginine, alanine, or glutamine produced mutant enzymes with apparent affinities for 3-P-glycerate 10-160-fold lower than that of the wild-type enzyme. The glutamic acid mutant enzyme was inhibited by 3-P-glycerate. These mutations had lesser impact on the kinetic constants for the substrates and inhibitor, P{sub i}, and on the thermal stability. These results indicate that both the charge and size of the residue at position 382 influence the binding of 3-P-glycerate. Site-directed mutagenesis was also performed to obtain a K382R-K419R double mutant. The apparent affinity for 3-P-glycerate of this double-mutant enzyme was 104-fold lower than that of the wild-type enzyme, and the specificity for activator of this mutant enzyme was altered. The K382R-K419R enzyme could not be phosphopyridoxylated, suggesting that other lysine residues aremore » not involved in the binding of 3-P-glycerate. 32 refs., 2 figs., 3 tabs.« less

Authors:
; ;  [1]
  1. Michigan State Univ., East Lansing, MI (United States)
Publication Date:
OSTI Identifier:
468992
Resource Type:
Journal Article
Journal Name:
Biochemistry (Eaton)
Additional Journal Information:
Journal Volume: 35; Journal Issue: 9; Other Information: PBD: 5 Mar 1996
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; LYSINE; MUTAGENESIS; PHOSPHOTRANSFERASES; AFFINITY; ARGININE; ENZYMES; SPECIFICITY; STRUCTURE-ACTIVITY RELATIONSHIPS; CYANOBACTERIA; ENZYME ACTIVITY

Citation Formats

Sheng, Jun, Charng, Yee-yung, and Preiss, J. Site-directed mutagenesis of Lysine{sup 382}, the activator-binding site, of ADP-Glucose pyrophosphorylase from Anabaena PCC 6120. United States: N. p., 1996. Web. doi:10.1021/bi952359j.
Sheng, Jun, Charng, Yee-yung, & Preiss, J. Site-directed mutagenesis of Lysine{sup 382}, the activator-binding site, of ADP-Glucose pyrophosphorylase from Anabaena PCC 6120. United States. doi:10.1021/bi952359j.
Sheng, Jun, Charng, Yee-yung, and Preiss, J. Tue . "Site-directed mutagenesis of Lysine{sup 382}, the activator-binding site, of ADP-Glucose pyrophosphorylase from Anabaena PCC 6120". United States. doi:10.1021/bi952359j.
@article{osti_468992,
title = {Site-directed mutagenesis of Lysine{sup 382}, the activator-binding site, of ADP-Glucose pyrophosphorylase from Anabaena PCC 6120},
author = {Sheng, Jun and Charng, Yee-yung and Preiss, J.},
abstractNote = {Previous studies have shown that a highly conserved lysyl residue (Lys{sup 419}) near the C-terminus of Anabaena ADP-glucose pyrophosphorylase is involved in the binding of 3-P-glycerate, the allosteric activator. Phosphopyridoxylation of the K419R mutant enzyme modified another conserved lysyl residue (Lys{sup 382}), suggesting that this residue might be also located within the activator-binding site. Site-directed mutagenesis of Lys{sup 382} of the Anabaena enzyme was performed to determine the role of this residue. Replacing Lys{sup 382} with either arginine, alanine, or glutamine produced mutant enzymes with apparent affinities for 3-P-glycerate 10-160-fold lower than that of the wild-type enzyme. The glutamic acid mutant enzyme was inhibited by 3-P-glycerate. These mutations had lesser impact on the kinetic constants for the substrates and inhibitor, P{sub i}, and on the thermal stability. These results indicate that both the charge and size of the residue at position 382 influence the binding of 3-P-glycerate. Site-directed mutagenesis was also performed to obtain a K382R-K419R double mutant. The apparent affinity for 3-P-glycerate of this double-mutant enzyme was 104-fold lower than that of the wild-type enzyme, and the specificity for activator of this mutant enzyme was altered. The K382R-K419R enzyme could not be phosphopyridoxylated, suggesting that other lysine residues are not involved in the binding of 3-P-glycerate. 32 refs., 2 figs., 3 tabs.},
doi = {10.1021/bi952359j},
journal = {Biochemistry (Eaton)},
number = 9,
volume = 35,
place = {United States},
year = {1996},
month = {3}
}