Mechanistic studies of microbial ATP sulfurylases and phosphomevalonate kinase
Pilloff, Daniel Eric
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Phosphorylation and sulfation modifications rank among the most prevalent forms of biochemical signaling. These are important examples of the much larger classes of phospho- and sulfoester-reactions that constitute the vast majority of the cell's metabolism. The bulk of this thesis describes mutagenesis studies of the sulfate-activating ATP sulfurylase systems from Escherichia coli and Saccharomyces cerevisiae, and the remainder reports on the determination of the steady-state mechanism of phosphomevalonate kinase from Streptococcus pneumoniae.;The E. coli ATP sulfurylase system is composed of two separate subunits, a CysD adenylyltransferase and a CysN GTPase, which assemble to form a tetramer of heterodimers. The free energy of GTP hydrolysis is used to drive the formation of activated sulfate. Four highly conserved residues---serine 35, glycine 37, aspartate 39, and serine 40---of the pyrophosphate-binding region of the phosphoadenylyltransferase active-site were individually mutagenized to alanine to probe their contributions to catalysis. Gly 37, Asp 39, and Ser 40 were found to be kinetically-important components of the pyrophosphate-binding region, while Ser 35 did not contribute significantly to PPi-binding interactions. Residues of the GTPase active site were also mutated to probe their contributions to catalysis. The P-loop lysine 40, Switch 1 threonine 94, and Switch II histidine 117 residues were all individually essential to the catalysis.;The native S. cerevisiae ATP sulfurylase system is a hexamer of monomeric phosphoadenylyltransferases. The function of the non-catalytic APS kinase-resembling domain located at the C-terminus of the S. cerevisiae ATP sulfurylase was studied by comparing the activity of native and C-terminal-truncated forms of the enzyme. The steady-state Michaelis constants for the two forms of the enzyme were similar. However, the truncated form of the enzyme was not able to self-organize into the hexameric native-like assembly.;The S. pneumoniae phosphomevalonate kinase was observed to have a random binding mechanism in both directions, and the steady-state Michaelis constants for the reactions were measured. This work also reports the initial observation of apparent inhibition of the mevalonate kinase by phosphomevalonate, which has been more fully characterized by other members of our group.