Abstract
We have shown that lysine-193 of the conserved K-T-G-G motif, functionally different from its counterpart in E.coli glycogen synthase, is involved in catalysis rather than ADP-glucose binding of maize (Zea mays L.) starch synthase Ha (Gao et al., 2001). To further study involvement of lysine residue(s) in catalysis and ADP-glucose binding of maize SSHa, three conserved lysine residues—lysine-465, lysine-469, and lysine-497—were subjected to site-directed mutagenesis. Kinetic characterization showed that mutations at lysine-497 dramatically increased the Km for ADP-glucose, and moreover K497Q, K497N and K497E increased the Km for ADP-glucose more than did mutation K497H. This result strongly suggests that lysine-497 is mainly involved in substrate ADP-glucose binding, with the positive charge on the side chain at position 497 being important to ADP-glucose binding. Replacement of glutamine or glutamic acid in lysine-465 and lysine-469 resulted in complete loss of activity, whereas the conservative replacement K465R and K469R decreased activity but had no influence on ADP-glucose binding. A simulated threedimensional protein structure model has been proposed and discussed for the possible mechanism of maize SSHa substrate ADP-glucose binding and catalysis.
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