SULFATE ADENYLATION-BIOCHEMISTRY & ENZYMOLOGY

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DESCRIPTION(adapted from applicant's abstract): ATP sulfurylase, from E. coli, catalyzes and conformationally couples the free energies of GTP hydrolysis and activated sulfate synthesis (reactions 1 and 2). The enzyme is the only example of a GTPase/target complex that couples the free energy of two chemical reactions. During the catalytic cycle, allosteric interactions between the active sites drive structural changes that control the progression of the individual reactions. These linking events result in an interdependence that fixes the stoichiometry of the reactions, and couples their free energies. The proposed studies are focused on these linking events and will establish paradigms for the allosteric interactions that occur in GTPase/target complexes, and the conformational coupling of free energy. ATP + SO(4) <-> APS + PPi (1) GTP + H(2)O <-> GDP + Pi + H(+) (2) In the second and final step of the sulfate activation pathway, the gamma-phosphoryl group of ATP is transferred to the 3'-hydroxyl of APS to form PAPS (reaction 3). ATP + APS <-> PAPS + ADP (3) PAPS is the only known sulfuryl group donor in metabolism. Sulfuryl transfer, much like phosphoryl transfer, is used extensively by the cell to regulate metabolite activity. In humans, the entire sulfate activating pathway is contained in a single enzyme, PAPS synthetase. This monomeric 73 kDa polypeptide catalyzes reactions 1 and 3 and channels APS between its active-sites. The human PAPS synthetase has been expressed and purified to homogeneity in the principal investigator's laboratory. Structures of channeling systems are rare and have revealed the amazing architecture that these enzymes use to shunt substrates between their active sites. PAPS synthetase promises to be one of those systems. Crystals of PAPS synthetase have been obtained that diffract to 2.8 angstroms. The existing paradigms suggest that channeling will require extensive allosteric interactions between active sites of PAPS synthetase. Dr. Leyh will initiate a structural and functional characterization of this fascinating system.