AAA-AT recognizes both AAA and Glu as amino donors with a decreased Km value of Glu ( Table 3); however, negligible activity was observed for Asp, which is one methylene unit shorter than Glu. When kinetic analysis was performed for Asp at a fixed concentration (1 mM) of 2-OG, the Km value for Asp and the kcat value for the reaction were determined to be 381 ± 61 mM and 0.046 ± 0.005 s?1, respectively. Arg23 from α2 helix recognizes the side-chain carboxylates of PPE and PPA at almost the same position. The markedly larger Km value of Asp, 820 and 470-fold larger than those of Glu and AAA, respectively, suggests that
PF-00562271 Arg23 cannot bind the β-carboxyl group of Asp tightly. Therefore, it is suggested that although α2 helix is highly flexible to accept various amino acids, no further dislocation of the helix suitable for binding the shorter side chain of Asp is allowed. Furthermore, considering that recognition by Arg23 of the γ-carboxyl group of the Glu moiety of PPE dislocates the Cα
atom of the Glu moiety as described above, we presume that the Cα atom of Asp must be located apart from the position suitable for the reaction even when Asp is bound to AAA-AT.Table 3. Kinetic parameters of AAA-AT variants.EnzymeSubstrateKm (mM)kcat (s?1)kcat/Km (M?1 s?1)
