Thermodynamics of an aminoglycoside modifying enzyme with low substrate promiscuity: The aminoglycoside N3 acetyltransferase-VIa

Kinetic, thermodynamic, and structural properties of the aminoglycoside N3-acetyltransferase-VIa (AAC-VIa) are determined. Among the aminoglycoside N3-acetyltransferases, AAC-VIa 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 k_cat values. Larger differences in K_M (∼40-fold) resulted in ∼30-fold variation in k_cat/K_M. Binding of aminoglycosides to AAC-VIa was enthalpically favored and entropically disfavored with a net result of favorable Gibbs energy (ΔG < 0). 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 N3-acetyltransferases, where ligand binding causes more protonation. The change in heat capacity (ΔCp) was different in H₂O and D₂O for the binary enzyme–sisomicin complex but remained the same in both solvents for the ternary enzyme–CoASH–sisomicin complex. Unlike, most other aminoglycoside-modifying enzymes, the values of ΔCp were within the expected range of protein-carbohydrate interactions. Solution behavior of AAC-VIa was also different from the more promiscuous aminoglycoside N3-acetyltransferases and showed a monomer-dimer 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-protein interactions and differences in protein behavior in solution provide few clues on the limited substrate profile of this enzyme despite its >55% sequence similarity to the highly promiscuous aminoglycoside N3-acetyltransferase. Proteins 2017; 85:1258–1265.