Metal Binding Network of Pigeon Liver Malic Enzyme

• Main Authors: Chen Yen-I, 陳嬿伊
• Other Authors: Chang Gu-Chang
• Format: Others
• Language: en_US
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/37659984002313402809

碩士 === 國防醫學院 === 生物化學研究所 === 89 === Pigeon liver malic enzyme catalyzes the divalent metal ion (Mn2+ or Mg2+) dependent reversible oxidative decarboxylation of L-malate. The enzymatic reaction proceeds in two consecutive steps. L-malate is first oxidized by NADP+ generating an enzyme-bound oxaloacetate, which is then decarboxylated to give CO2 and pyruvate. The enzyme is exised as a tetramer in solution, which has square-planner arrangement of the four monomers. The previously studies in our lab show 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. Examining the resolved crystal structure of the pigeon liver malic enzyme, Asp141 is not a direct metal binding ligand. The side chain of Asp141 is located next to Phe236, which directly follows the Mn2+ ligands Glu234 and Asp235. The distance of the side chains between Asp141 and Phe236 in the pigeon liver malic enzyme is approximately 3.7 Å. 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. In this study, I used the “double mutant cycle” method to study the possible interaction between these two residues. Because Asp141 and Phe236 are highly conserved in most species of malic enzyme, I have made various mutants of these two residues. I cloned and expressed eight mutants and generated four double mutant cycles. Initial velocity experiments were performed to derive the various kinetic parameters, which can be used to analysis the free energy change and coupling energy. The coupling energy (△△Gint) for the double mutant cycles were approximately 1.64 kcal/mol, showing a Van der Waals interaction between Asp141 and Phe236. Combine the kinetic data, thermodynamic data, and structure information, I suggest that there is a Van der Waals interaction between the two residues. Through this kind of interaction, Asp141 was involved in the Mn2+-L-malate binding of the pigeon liver malic enzyme.