Although Ser20 does not recognize AAA and Glu directly, replacement of Ser20 altered the enzyme to exhibit parameters similar to those of Arg23 mutant. In PPA and PPE complexes, Ser20 forms a
A-803467 with the main-chain amide of Arg23 by its hydroxyl group (Fig. 3A). We speculated that Ser20 might stabilize the Arg23-N location, which allows Arg23 to have tight ionic interactions with acidic substrates. In the PPL complex, Ser20 is hydrogen-bounded with Gln261? from another subunit (Fig. 4A). In this complex, neighboring Glu257? forms a hydrogen bond with Arg23. In both cases, Ser20 also plays a crucial role in placing the substrates at appropriate position in the active site indirectly. When the substrate is bound, α2 helix occupies similar position to cover the active site, forming a closed form, whereas in the PLP complex without substrate, the helix is displaced 5–6 ? to open the active site cleft (open form) (Fig. 4C). Thus, AAA-AT adopts closed or open form in the presence or the absence of substrate. The crystal structures and site-directed mutagenesis revealed
simple leaf the conserved pair of Arg23 and Ser20 plays important roles in not only direct binding of AAA and Glu but also closed form formation in AAA-AT.
