Identification the acidic pKa in the pH-rate profile of pigeon NADP-dependent malic enzyme

• Main Authors: Cheng, Wan-Ju, 鄭婉如
• Other Authors: 周慰遠
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/51658377150127801132

碩士 === 國防醫學院 === 生物化學研究所 === 95 === NADP+-dependent malic enzyme follows a general acid-base mechanism to catalyze oxidative decarboxylation of L-malate to pyruvate in the presence of divalent metal ion. This reaction involved hydride transfer, decarboxylation and tautomerization three steps. As a general acid-base mechanism, the pH-rate profile of the wild-type enzyme showed a bell-shaped pattern, in which the acidic and basic pK values are derived from the general base and general acid residues, respectively. Based on our previous site-directed mutagensis, kinetic studies, and chemical rescues studies, the D258 residue was proposed as a general base to facilitate the hydride transfer and decraboxylation reactions. However, a bell-shaped pH-rate profile of D258A mutant, which is the same as that of wild-type enzyme, is obtained. To identify the chemical group responsible for the acidic site pKa, different metal ions were used to determine the pH dependence of the kinetic parameters for malate oxidoreduction and decarboxylation reaction. The acidic pKa values of pH-rate profiles are in the order of Cd2+ > Mg2+ > Mn2+ > Co2+, which is consistent with the order of the values of pKa of the metal-bound hydroxyl group. The pH-rate profile study of the reversed reaction, in which pyruvate lacking the β-hydroxyl group is the substrate, was performed. A bell-shaped pH-rate profile was obtained for the wild-type enzyme. The basic pKb values were independent to the metal ions used in the reversed reaction. The kcat values of D258A mutant were pH-independent at basic site. These results suggested that the pKa observed in the bell-shaped pH-rate profile of wild-type is derived from both the deprotonation of the β-hydroxyl group of malate and the side chain of D258 residue which is the general base in the catalytic reaction.