Studies on the Roles of Glutamyl and Aspartyl Residues of Pigeon Liver Malic Enzyme by Site-Directed Mutagenesis

• Main Authors: Chen Yu-jung, 陳昱蓉
• Other Authors: Chou Wei-yuan
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/26250448854048515881

碩士 === 國防醫學院 === 生物化學研究所 === 86 === Pigeon liver malic enzyme(EC 1.1.1.40) catalyzed the oxidative decarb oxylation of L-malate to CO2 and pyruvate, with concomitant reduction of NADP+to NADPH. Based on the chemical modification studies, the carboxyl group of g lutamate and aspartate residues may participate in acid-base catalytic reactions. Twenty-seven glutamate and aspartate were identified as highly conserved amino acid residues among several species. All these were mutated by the alanine scanning mutagenesis except Asp258, which was identified as one of the divalent metal binding site. The preliminary kinetic studies of these mutants revealed that the apparent KmMg and KmMalate values for E234A were increased by about 610-fold and 35-fold, respectively. The kcat(app) value for D235A was decreased to about 0.5% of that of the wild type. The apparent KmNADP value for D324A was increased by 41-fold.From the initial velocity experiment, the KmMg and KdMg of E234A were increased by 48-fold and 87-fold. The specific constant (kcat/KdMg) of E234A mutant was 1.53% and its catalytic efficiency (kcat/KmN ADP(app)．KdMalate．KdMg) was only 0.0036%. The E234A mutant was also insensi tive to metal2+-ascorbate inactivation and cleavage. We proposed that E234A may be one of the divalent metal chelate sites of the malic enzyme.Different from the wild type, D235A mutant can not inactivated by carboxyl specific Woodward*s reagent K (WRK) modification. The Km(app) values for D235E, D235N, D235V for L-malate, Mn2+, NADP+ remained unchanged, but the kcat(app) values were decreased to 1-7%. For reduction partial reaction, the kcat(app) value was reduced to 0.5% and the KmPyruvate(app) value was decreased by a factor of 0.09. For decarboxylation partial reaction, the kcat(app) value of D235A was reduced to 1%. These results showed that Asp235 may participate in the catalytic reaction of pigeon liver malic enzyme.Based on kinetic studies, the apparent KmNADP(app) value of D324A and D324E were increased 41-fold and 29-fold to wild type, respectively. The D324A mutant showed the KmPyruvate(app) value similar to that of the wild type. The kcat(app) value of D324A was decreased to 15% of that of wild type in the reduction of pyruvate to lactate. D324A was also unable to bind to 2*,5*-ADP agarose during the protein purification. These resu lts suggest that Asp324 may either involve in nucleotide binding directly or is located closely to the active site.