2jev: Difference between revisions
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==Overview== | ==Overview== | ||
The N1-acetylation of spermidine and spermine by spermidine/spermine, acetyltransferase (SSAT) is a crucial step in the regulation of the, cellular polyamine levels in eukaryotic cells. Altered polyamine levels, are associated with a variety of cancers as well as other diseases, and, key enzymes in the polyamine pathway, including SSAT, are being explored, as potential therapeutic drug targets. We have expressed and purified, human SSAT in Escherichia coli and characterized its kinetic and chemical, mechanism. Initial velocity and inhibition studies support a random, sequential mechanism for the enzyme. The bisubstrate analogue, N1-spermine-acetyl-coenzyme A, exhibited linear, competitive inhibition, against both substrates with a true Ki of 6 nM. The pH-activity profile, was bell-shaped, depending on the ionization state of two groups, exhibiting apparent pKa values of 7.27 and 8.87. The three-dimensional, crystal structure of SSAT with bound bisubstrate inhibitor was determined, at 2.3 A resolution. The structure of the SSAT-spermine-acetyl-coenzyme A, complex suggested that Tyr140 acts as general acid and Glu92, through one, or more water molecules, acts as the general base during catalysis. On the, basis of kinetic properties, pH dependence, and structural information, we, propose an acid/base-assisted reaction catalyzed by SSAT, involving a, ternary complex. | The N1-acetylation of spermidine and spermine by spermidine/spermine, acetyltransferase (SSAT) is a crucial step in the regulation of the, cellular polyamine levels in eukaryotic cells. Altered polyamine levels, are associated with a variety of cancers as well as other diseases, and, key enzymes in the polyamine pathway, including SSAT, are being explored, as potential therapeutic drug targets. We have expressed and purified, human SSAT in Escherichia coli and characterized its kinetic and chemical, mechanism. Initial velocity and inhibition studies support a random, sequential mechanism for the enzyme. The bisubstrate analogue, N1-spermine-acetyl-coenzyme A, exhibited linear, competitive inhibition, against both substrates with a true Ki of 6 nM. The pH-activity profile, was bell-shaped, depending on the ionization state of two groups, exhibiting apparent pKa values of 7.27 and 8.87. The three-dimensional, crystal structure of SSAT with bound bisubstrate inhibitor was determined, at 2.3 A resolution. The structure of the SSAT-spermine-acetyl-coenzyme A, complex suggested that Tyr140 acts as general acid and Glu92, through one, or more water molecules, acts as the general base during catalysis. On the, basis of kinetic properties, pH dependence, and structural information, we, propose an acid/base-assisted reaction catalyzed by SSAT, involving a, ternary complex. | ||
==Disease== | |||
Known disease associated with this structure: Keratosis follicularis spinulosa decalvans OMIM:[[http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=313020 313020]] | |||
==About this Structure== | ==About this Structure== | ||
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[[Category: transferase]] | [[Category: transferase]] | ||
''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 12 22:54:45 2007'' | ||
