title: Redox-linked gating of nucleotide binding by the N-terminal domain of adenosine 5'-phosphosulfate kinase.
abstract: Adenosine 5'-phosphosulfate kinase (APSK) catalyzes the phosphorylation of adenosine 5'-phosphosulfate (APS) to 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Crystallographic studies of APSK from Arabidopsis thaliana revealed the presence of a regulatory intersubunit disulfide bond (Cys(86)-Cys(119)). The reduced enzyme displayed improved catalytic efficiency and decreased effectiveness of substrate inhibition by APS compared with the oxidized form. Here we examine the effect of disulfide formation and the role of the N-terminal domain on nucleotide binding using isothermal titration calorimetry (ITC) and steady-state kinetics. Formation of the disulfide bond in A. thaliana APSK (AtAPSK) inverts the binding affinities at the ATP/ADP and APS/PAPS sites from those observed in the reduced enzyme, consistent with initial binding of APS as inhibitory, and suggests a role for the N-terminal domain in guiding nucleotide binding order. To test this, an N-terminal truncation variant (AtAPSKΔ96) was generated. The resulting protein was completely insensitive to substrate inhibition by APS. ITC analysis of AtAPSKΔ96 showed decreased affinity for APS binding, although the N-terminal domain does not directly interact with this ligand. Moreover, AtAPSKΔ96 displayed reduced affinity for ADP, which corresponds to a loss of substrate inhibition by formation of an E·ADP·APS dead end complex. Examination of the AtAPSK crystal structure suggested Arg(93) as important for positioning of the N-terminal domain. ITC and kinetic analysis of the R93A mutant also showed a complete loss of substrate inhibition and altered nucleotide binding affinities, which mimics the effect of the N-terminal deletion. These results show how thiol-linked changes in AtAPSK alter the energetics of binding equilibria to control its activity.
authors: Department of Biology, Washington University, St Louis, Missouri 63130, USA.
authors: Geoffrey E
authors: GE
authors: Ravilious
authors: Corey S
authors: CS
authors: Westfall
authors: Joseph M
authors: JM
authors: Jez
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins; D002384:Catalysis; D007700:Kinetics; D010084:Oxidation-Reduction; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D013438:Sulfhydryl Compounds; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase