Characterization of the interaction between Asp141 and Phe236 in the Mn2+-L-malate binding of the pigeon liver malic enzyme

         

YEN-I CHEN AND GU-GANG CHANG

Department of Biochemistry, National Defense Medical Center

 

We have previously reported that the pigeon liver malic enzyme was inactivated by the Fe2+-ascorbate or Cu2+-ascorbate system. The enzyme was cleaved at several positions, which supposed to be the metal binging site. Asp141 is one of these cleavage sites. Involvement of  Asp141 in the Mn2+-L-malate binding for pigeon liver malic enzyme was also suggested by site-specific mutagenesis. However, the X-ray crystal structure of the human mitochondrial malic enzyme did not support Asp141 as a direct metal binding ligand. Examining the resolved crystal structure of human mitochondrial malic enzyme , Asp141 is located next to Phe236, which directly follows the Mn2+ ligands Glu234 and Asp235. Thus, a local change in the Asp141 might, through Phe236, perturb the conformation of Glu234 and Asp235 and have an indirect effect on the Mn2+ binding. Because Asp141 and Phe236 are highly conserved in most species of malic enzyme, we used a double mutant cycle to study the possible interaction between these two residues. The dissociation constant (Kd) for Mn2+ of the D141A and F236A mutants were respectively increased by approximately 6 and 65 fold compared to the wild type. However the Kd,Mn for the double mutant D141A/F236A were only increased by 150 fold. The coupling energy (△△Gint) calculated from the double mutant cycle was negative, indicating an antagonistic effect between these two residues. Such antagonistic effect indicates that cooperativity in Mn2+ binding did exist between Asp141 and Phe236 residues.

 

 

 

 

Note: