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Among the aminoglycoside <jats:italic>N3<\/jats:italic>\u2010acetyltransferases, AAC\u2010VIa has one of the most limited substrate profiles. Kinetic studies showed that only five aminoglycosides are substrates for this enzyme with a range of fourfold difference in <jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub> values. Larger differences in <jats:italic>K<\/jats:italic><jats:sub>M<\/jats:sub> (\u223c40\u2010fold) resulted in \u223c30\u2010fold variation in <jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub>\/<jats:italic>K<\/jats:italic><jats:sub>M<\/jats:sub>. Binding of aminoglycosides to AAC\u2010VIa was enthalpically favored and entropically disfavored with a net result of favorable Gibbs energy (\u0394<jats:italic>G<\/jats:italic>\u2009&lt;\u20090). A net deprotonation of the enzyme, ligand, or both accompanied the formation of binary and ternary complexes. This is opposite of what was observed with several other aminoglycoside <jats:italic>N3<\/jats:italic>\u2010acetyltransferases, where ligand binding causes more protonation. The change in heat capacity (\u0394Cp) was different in H<jats:sub>2<\/jats:sub>O and D<jats:sub>2<\/jats:sub>O for the binary enzyme\u2013sisomicin complex but remained the same in both solvents for the ternary enzyme\u2013CoASH\u2013sisomicin complex. Unlike, most other aminoglycoside\u2010modifying enzymes, the values of \u0394Cp were within the expected range of protein\u2010carbohydrate interactions. Solution behavior of AAC\u2010VIa was also different from the more promiscuous aminoglycoside <jats:italic>N3<\/jats:italic>\u2010acetyltransferases and showed a monomer\u2010dimer equilibrium as detected by analytical ultracentrifugation (AUC). Binding of ligands shifted the enzyme to monomeric state. Data also showed that polar interactions were the most dominant factor in dimer formation. Overall, thermodynamics of ligand\u2010protein interactions and differences in protein behavior in solution provide few clues on the limited substrate profile of this enzyme despite its &gt;55% sequence similarity to the highly promiscuous aminoglycoside <jats:italic>N3<\/jats:italic>\u2010acetyltransferase. Proteins 2017; 85:1258\u20131265. \u00a9 2017 Wiley Periodicals, Inc.<\/jats:p>","DOI":"10.1002\/prot.25286","type":"journal-article","created":{"date-parts":[[2017,3,19]],"date-time":"2017-03-19T05:54:30Z","timestamp":1489902870000},"page":"1258-1265","source":"Crossref","is-referenced-by-count":8,"title":["Thermodynamics of an aminoglycoside modifying enzyme with low substrate promiscuity: The aminoglycoside <i>N3<\/i> acetyltransferase\u2010VIa"],"prefix":"10.1002","volume":"85","author":[{"given":"Prashasti","family":"Kumar","sequence":"first","affiliation":[{"name":"Graduate School of Genome Science and Technology The University of Tennessee and Oak Ridge National Laboratory Knoxville Tennessee 37996"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1420-455X","authenticated-orcid":false,"given":"Engin H.","family":"Serpersu","sequence":"additional","affiliation":[{"name":"Graduate School of Genome Science and Technology The University of Tennessee and Oak Ridge National Laboratory Knoxville Tennessee 37996"},{"name":"Department of Biochemistry, Cellular and Molecular Biology The University of Tennessee Knoxville Tennessee 37996"},{"name":"National Science Foundation Arlington Virgina 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