Structural mechanism for substrate inhibition of the adenosine 5'-phosphosulfate kinase domain of human 3'-phosphoadenosine 5'-phosphosulfate synthetase 1 and its ramifications for enzyme regulation.
In mammals, the universal sulfuryl group donor molecule 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is synthesized in two steps by a bifunctional enzyme called PAPS synthetase. The APS kinase domain of PAPS synthetase catalyzes the second step in which APS, the product of the ATP-sulfurylase domain, is phosphorylated on its 3'-hydroxyl group to yield PAPS. The substrate APS acts as a strong uncompetitive inhibitor of the APS kinase reaction. We generated truncated and point mutants of the APS kinase domain that are active but devoid of substrate inhibition. Structural analysis of these mutant enzymes reveals the intrasubunit rearrangements that occur upon substrate binding. We also observe intersubunit rearrangements in this dimeric enzyme that result in asymmetry between the two monomers. Our work elucidates the structural elements required for the ability of the substrate APS to inhibit the reaction at micromolar concentrations. Because the ATP-sulfurylase domain of PAPS synthetase influences these elements in the APS kinase domain, we propose that this could be a communication mechanism between the two domains of the bifunctional enzyme.
Sekulic
Nikolina
N
Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, IL 60607, USA.
Konrad
Manfred
M
Lavie
Arnon
A
D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D006706:Homeostasis; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D016296:Mutagenesis; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D017384:Sequence Deletion; D013430:Sulfate Adenylyltransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase; C022322:PAPS synthetase; D013430:Sulfate Adenylyltransferase