Structure and Function of Amino Acid Ammonia-lyases

proton from the 3-position of the substrate. On the basis of this analysis, the likely mechanism for MAL involves abstraction of the 3-proton of the substrate by Lys331, stabilization of the enolic intermediate by the metal ion and possibly His194, and collapse of the intermediate with elimination of ammonia to give mesaconic acid (Fig. 7). When the primary structures of MAL and enzymes of the enolase superfamily are compared, there is only very TABLE IV Structure of MAL. 1. Each subunit consists of two domains (1 /160 aa, 170 /411 aa) linked by a single extended b-type structure 2. Domain I: (1 /160 aa) a three-stranded antiparallel b sheet and an antiparallel, four a helix bundle 3. Domain II: (170 /411 aa) an eight-stranded TIM barrel structure 4. Active site: Mg2 is octahedrally coordinated by one carboxyl oxygen of Asp238, Glu273, and Asp307, two H2O, which are both linked to the carboxyl of Glu308, and one of the carboxyl oxygens of the substrate 5. The dimer appears to be formed by interaction between the Cterminal helix of the TIM barrel domain FIGURE 5 Coordination of Mg with one carboxyl oxygen of Asp238, Glu273, Asp307, and methylaspartic acid, and two H2O linked to Glu308 (Levy et al. , 2002). FIGURE 6 Electron density for the bound substrate, 3-methylaspartic acid with Mg (Levy et al. , 2002). 136 Y. ASANO et al.