Structural and functional studies of sulfate activating enzymes

This dissertation summarizes the current efforts to understand the sulfate activation pathway. This pathway contains the enzymes ATP sulfurylase and APS kinase.;Chapter 2 describes the kinetic and structural analysis of a twenty-two amino acid truncation mutant of APS kinase from Penicillium chrysogenum. Kinetic data show that the mutant enzyme, which lacks a short N-terminal helix, is dramatically less active than the wild type enzyme and also does not display substrate inhibition with respect to APS, a trait common to all known wild type APS kinases. The 2.50 A structural data reveal that the mutant enzyme experiences small shifts in three rigid bodies. These shifts lead to a more open (P)APS binding site in one monomer and help explain the lack of substrate inhibition.;Chapter 3 gives details of the kinetic characterization of ATP sulfurylase and APS kinase from the chemolithotrophic bacteria Thiobacillus denitrificans. ATP sulfurylase displays the most activity for enzymes of its kind and is optimized for the ATP synthesis direction. APS kinase displays substrate inhibition with respect to APS, but has a higher optimal APS concentration than APS kinases from sulfate assimilators. This work also shows that a putative ATP sulfurylase domain at the N-terminus of APS kinase is an inactive domain that is involved in substrate inhibition.;Chapter 4 presents the crystal structure of APS kinase from Thiobacillus denitrificans at 2.95 A resolution. The asymmetric unit is comprised of three monomers, but the biologically relevant hexamer is generated through crystallographic symmetry. The APS kinase monomer is much larger than that from other organism. This is due to the large inactive N-terminal ATP sulfurylase-like domain. The structure reveals numerous differences in the ATP-sulfurylase like domain compared to its active counterparts.;Chapter 1 reviews background information regarding sulfur metabolism in multiple types of organisms as well research that has established the current understanding of ATP sulfurylase and APS kinase.;Chapter 5 summarizes numerous future studies that still need to be addressed. This chapter also shows comparisons of kinetic constants for ATP sulfurylase and APS kinase from multiple organisms.