Nucleotide binding site communication in Arabidopsis thaliana adenosine 5'-phosphosulfate kinase.
Adenosine 5'-phosphosulfate kinase (APSK) catalyzes the ATP-dependent synthesis of adenosine 3'-phosphate 5'-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 Asp(136), 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.
Department of Biology, Washington University, St. Louis, MO 63130, USA.
Geoffrey E
GE
Ravilious
Joseph M
JM
Jez
D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D008956:Models, Chemical; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism
D000244:Adenosine Diphosphate; D013455:Sulfur; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase