ATP-Binding Site

The binding site for ATP and its associated magnesium ions in APH(30)-IIIa is located in the deep cleft between the N- and C-terminal lobes. Of the four absolutely conserved residues in both eukaryotic protein kinases and aminoglycoside phosphotransferases, three are located in the ATP-binding pocket where they interact with the cofactor and its associated magnesium ions—Asp190, Asn195 and Asp208 (Fig. 3). Two additional highly conserved residues, Lys44 and Glu60, are also found in the active site.

Fig. 3 The nucleotide-binding pocket of APH(3')-IIIa. The backbone is shown as ribbon and conserved residues are drawn as balls-and-sticks, both in light grey. ATP and its associated magnesium ions are drawn as balls-and-sticks in black

Lys44 is positioned over the binding site, interacting with the a- and b-phosphates of the cofactor. Evidence from mutagenesis studies suggests that Lys44 influences the Km for ATP and thus makes an important contribution to ATP binding (Hon et al. 1997). The analogous lysine residue in the protein kinase family is also positioned to interact with the a- and b-phosphates of ATP. Glu60 is positioned in such a way that it hydrogen bonds to the side chain of Lys44, orienting it so that it interacts with the a- and b-phosphates of ATP.

In protein kinases, the residue corresponding to Asp190 has been proposed as a general base assisting in substrate deprotonation (Madhusudan et al. 1994). Although mutagenesis of Asp190 in APH(3')-IIIa results in drastically lower activity, supporting a role for this residue in catalysis, its role in catalysis has not been definitively identified (Hon et al. 1997; Zhou and Adams 1997). The function of Asp190 may be limited to positioning of the substrate hydroxyl group during phosphoryl transfer (Boehr et al. 2001b).

Mutagenesis studies of Asn195 indicated that this residue was important for ATP binding. Since Asn195 interacts with ATP via a magnesium ion, it has been suggested that the decrease in ATP affinity is the result of a non-optimally coordinated metal ion (Boehr et al. 2001b). Asp208 is a ligand of both active site metal ions, and an Asp208Ala mutation results in a protein without detectable aminoglycoside kinase activity (Boehr et al. 2001b). Thus, Asp208 appears to be critical for catalysis in APH(3')-IIIa, facilitating the generation or stabilization of the transition state.

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