Abstract
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.
Highlights
Substrate or product inhibition phenomena are widely found in nature and believed to be important for metabolic feedback regulation processes [1,2,3]
APS kinase is an evolutionarily conserved enzyme catalyzing the second step in the formation of 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS), the universal sulfuryl donor in biological systems
In the first reaction of PAPS formation, inorganic sulfate is converted to APS by ATP-sulfurylase
Summary
Substrate or product inhibition phenomena are widely found in nature and believed to be important for metabolic feedback regulation processes [1,2,3]. One monvation of the N-terminal region in APS kinase domains is low, omer has both nucleotides bound, as seen before, whereas the two residues are highly conserved: an invariant asparagine at second monomer has only dADP present
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