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Title: Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5;-Phosphosulfate Kinase

Abstract

Adenosine 5{prime}-phosphosulfate kinase (APSK) catalyzes the ATP-dependent synthesis of adenosine 3{prime}-phosphate 5{prime}-phosphosulfate (PAPS), which is an essential metabolite for sulfur assimilation in prokaryotes and eukaryotes. Using APSK from Arabidopsis thaliana, we examine the energetics of nucleotide binary and ternary complex formation and probe active site features that coordinate the order of ligand addition. Calorimetric analysis shows that binding can occur first at either nucleotide site, but that initial interaction at the ATP/ADP site was favored and enhanced affinity for APS in the second site by 50-fold. The thermodynamics of the two possible binding models (i.e. ATP first versus APS first) differs and implies that active site structural changes guide the order of nucleotide addition. The ligand binding analysis also supports an earlier suggestion of intermolecular interactions in the dimeric APSK structure. Crystallographic, site-directed mutagenesis, and energetic analyses of oxyanion recognition by the P-loop in the ATP/ADP binding site and the role of Asp136, which bridges the ATP/ADP and APS/PAPS binding sites, suggest how the ordered nucleotide binding sequence and structural changes are dynamically coordinated for catalysis.

Authors:
;  [1]
  1. (WU)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Science Foundation (NSF)
OSTI Identifier:
1050080
Resource Type:
Journal Article
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 287; Journal Issue: 36; Journal ID: ISSN 0021-9258
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; ADENOSINE; AFFINITY; ARABIDOPSIS; CALORIMETRY; CATALYSIS; COMMUNICATIONS; ENZYMES; METABOLITES; MUTAGENESIS; NUCLEOTIDES; PHOSPHOTRANSFERASES; PROBES; PROTEIN STRUCTURE; SULFUR; SYNTHESIS; THERMODYNAMICS

Citation Formats

Ravilious, Geoffrey E., and Jez, Joseph M. Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5;-Phosphosulfate Kinase. United States: N. p., 2012. Web. doi:10.1074/jbc.M112.387001.
Ravilious, Geoffrey E., & Jez, Joseph M. Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5;-Phosphosulfate Kinase. United States. doi:10.1074/jbc.M112.387001.
Ravilious, Geoffrey E., and Jez, Joseph M. Fri . "Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5;-Phosphosulfate Kinase". United States. doi:10.1074/jbc.M112.387001.
@article{osti_1050080,
title = {Nucleotide Binding Site Communication in Arabidopsis thaliana Adenosine 5;-Phosphosulfate Kinase},
author = {Ravilious, Geoffrey E. and Jez, Joseph M.},
abstractNote = {Adenosine 5{prime}-phosphosulfate kinase (APSK) catalyzes the ATP-dependent synthesis of adenosine 3{prime}-phosphate 5{prime}-phosphosulfate (PAPS), which is an essential metabolite for sulfur assimilation in prokaryotes and eukaryotes. Using APSK from Arabidopsis thaliana, we examine the energetics of nucleotide binary and ternary complex formation and probe active site features that coordinate the order of ligand addition. Calorimetric analysis shows that binding can occur first at either nucleotide site, but that initial interaction at the ATP/ADP site was favored and enhanced affinity for APS in the second site by 50-fold. The thermodynamics of the two possible binding models (i.e. ATP first versus APS first) differs and implies that active site structural changes guide the order of nucleotide addition. The ligand binding analysis also supports an earlier suggestion of intermolecular interactions in the dimeric APSK structure. Crystallographic, site-directed mutagenesis, and energetic analyses of oxyanion recognition by the P-loop in the ATP/ADP binding site and the role of Asp136, which bridges the ATP/ADP and APS/PAPS binding sites, suggest how the ordered nucleotide binding sequence and structural changes are dynamically coordinated for catalysis.},
doi = {10.1074/jbc.M112.387001},
journal = {Journal of Biological Chemistry},
issn = {0021-9258},
number = 36,
volume = 287,
place = {United States},
year = {2012},
month = {8}
}