Ligand-Induced Structural Changes in Adenosine 5'-Phosphosulfate Kinase fromPenicillium chrysogenum†,‡

Eric B. Lansdon, Irwin H. Segel, Andrew J. Fisher
2002 Biochemistry  
Adenosine 5′-phosphosulfate (APS) kinase catalyzes the second reaction in the two-step, ATPdependent conversion of inorganic sulfate to 3′-phosphoadenosine 5′-phosphosulfate (PAPS). PAPS serves as the sulfuryl donor for the biosynthesis of all sulfate esters and also as a precursor of reduced sulfur biomolecules in many organisms. Previously, we determined the crystal structure of ligand-free APS kinase from the filamentous fungus, Penicillium chrysogenum [MacRae et al. (2000 ) Biochemistry 39,
more » ... ) Biochemistry 39, 1613-1621. That structure contained a protease-susceptible disordered region ("mobile lid"; residues 145-170). Addition of MgADP and APS, which together promote the formation of a nonproductive "dead-end" ternary complex, protected the lid from trypsin. This report presents the 1.43 Å resolution crystal structure of APS kinase with both ADP and APS bound at the active site and the 2.0 Å resolution structure of the enzyme with ADP alone bound. The mobile lid is ordered in both complexes and is shown to provide part of the binding site for APS. That site is formed primarily by the highly conserved Arg 66, Arg 80, and Phe 75 from the protein core and Phe 165 from the mobile lid. The two Phe residues straddle the adenine ring of bound APS. Arg 148, a completely conserved residue, is the only residue in the mobile lid that interacts directly with bound ADP. Ser 34, located in the apex of the P-loop, hydrogenbonds to the 3′-OH of APS, the phosphoryl transfer target. The structure of the binary E'ADP complex revealed further changes in the active site and N-terminal helix that occur upon the binding/release of (P)APS. Inorganic sulfate is converted into a biologically "activated" form, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), 1 via a two-step sequence catalyzed, in order, by the enzymes ATP sulfurylase (MgATP, sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (MgATP, APS 3′-phosphotransferase, EC 2.7.1.25): The sequences of APS kinases from over 30 different organisms show that this enzyme is highly conserved. In lower organisms, APS kinase exists as an independent enzyme, and the PAPS it produces is generally used for reductive sulfate assimilation (i.e., for the biosynthesis of cysteine, etc.). In mammals, the enzyme exists as part of a bifunctional "PAPS synthetase" that possesses both ATP sulfurylase and APS kinase activities (1-3). Mammals use PAPS solely for the production of sulfate esters, which play important roles in cellular homeostasis (4). The absence of one human isoform of PAPS synthetase has been associated with metastatic cell behavior in colon carcinoma cells (5). Most kinetics studies of APS kinase have focused on the enzymes from Penicillium chrysogenum (6-8), Escherichia coli (9-11), Arabidopsis thaliana (12, 13) and rat chondrosarcoma cells (14, 15) . APS kinase from the filamentous fungus P. chrysogenum is a homodimer, each 23.67 kDa monomer subunit containing 211 amino acid residues. Steady-state kinetics have shown that the enzyme follows a compulsory ordered mechanism in which MgATP binds before APS, and PAPS leaves before MgADP (6). APS can bind to E'MgADP, forming a catalytically inactive ("dead-end") ternary E'MgADP'APS complex. The formation of this complex is the basis of the substrate inhibition exhibited by APS (6, 16) . Substrate inhibition has also been reported for other APS kinases, including the closely related (sequence-wise) E. coli enzyme (9-11), but the kinetic mechanism of the bacterial enzyme †
doi:10.1021/bi026556b pmid:12427029 fatcat:fbo4bqfjwnblzo2noyzvpqkwau