The goal of this research is to study the biochemical processes involved in the adenylation of sulfate in E. coli K-12. Adenylated sulfate, or APS (adenosine 5'-phosphosulfate), is an obligate precursor in the biochemical assimilation of sulfate. The formation of APS is catalyzed by the enzyme ATP sulfurylase (ATP: sulfate adenyltransferase, EC 18.104.22.168) via reaction 1. ATP sulfurylase is ubiquitous in nature. Perhaps the most biomedically relevant role of adenylated sulfate in vertebrates is as the sulfuryl donor in sulfate transfer reactions. Sulfation is essential to the integrity of structural and connective tissues. Burgeoning research in the area of tyrosine sulfation has revealed subgroups of several protein classes, including extracellular proteins, hormones, neuropeptides, and proteins of the immune and blood clotting systems, which are sulfated at specific tyrosine residues. Decreased levels of APS have been correlated with limb deformity in mice, and with estrogen sulfation and the typing of primary breast tumors in man. A recently discovered protein (i.e. the effector) is required for stimulation of the forward rate of reaction 1. Interestingly, the effector has little or not influence on the rate of the reverse reaction. Thus, the effector presents an unusual form of metabolic regulation in that it can kinetically regulate the reversibility of the sulfate utilization pathway. Protein induced, unidirectional stimulation of enzyme activity is a somewhat rare phenomenon in biochemistry; characterization of the effector will serve as a paradigm for such processes. This project will purify, characterize, and clone the structural gene for the effector protein. Effector mediated alterations in the catalytic mechanism of ATP sulfurylase will be studied at the physical chemical, structure function level. The methods used in these studies will include steady state kinetics, single turnover kinetics, and nuclear and electron paramagnetic resonance.
|Effective start/end date
|9/2/95 → 3/31/14
- Biochemistry, Genetics and Molecular Biology(all)
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