W0186

Visualizing The Structural Changes in the Allosteric Inhibition of Fungal ATP Sulfurylase. Andrew J. Fisher^1,2, Ian J. MacRae², Eric B. Lansdon¹, and Irwin H. Segel², ¹Dept. of Chemistry and ²Section of Molecular and Cellular Biology, Univ. of California, Davis, CA 95616.

ATP sulfurylase is a ubiquitous enzyme involved in the activation and assimilation of sulfate. Sulfate activation proceeds in two steps. These are catalyzed, in order, by the enzymes ATP sulfurylase and APS kinase. The reactions produce the sulfonucleotides APS (adenosine 5'-phosphosulfate) and PAPS (3'-phosphoadenosine 5'-phosphosulfate):

MgATP + SO₄^2- MgPP_i + APS (ATP sulfurylase)

MgATP + APS PAPS + MgADP (APS kinase)

The fungal ATP sulfurylase enzyme is allosterically inhibited by PAPS, the product of the APS kinase reaction. The structures of the cooperative hexameric enzyme ATP sulfurylase from P. chrysogenum have been determined with the substrate APS bound, which represents the active R-state structure, and bound to its allosteric inhibitor PAPS representing the inhibited T-state of the enzyme. Comparison of the R-state structure to the T-state revealed that a large rotational rearrangement of domains occurs as a result of the R to T transition. The rearrangement is accompanied by the 17Å movement of a ten-residue loop out of the active site region resulting in an open, product-release-like structure of the catalytic domain. PAPS-binding is proposed to induce the allosteric transition by destabilizing an R-state-specific salt linkage between the N-terminal domain of one subunit and the allosteric domain of a trans-triad subunit. Disrupting this salt linkage by site-directed-mutagenesis induces cooperative inhibition behavior in the absence of allosteric effector confirming the role of this salt link.

Interestingly, the allosteric domain of ATP sulfurylase displays high sequence and structural homology to APS kinase. This suggests the possible evolution of fungal ATP sulfurylase from an ancestral bi-functional enzyme in which allosteric domain has retained only the (P)APS binding site for allosteric regulation but lost ATP binding and catalytic APS kinase activity.