Functional Roles of Asparagine 442 in the Open and Closed form Structure of Pigeon Liver Malic Enzyme

• Main Authors: Chiang,Hsuan-Hao, 江宣浩
• Other Authors: Chou,Wei-Yuan
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/14191557904210856232

碩士 === 國防醫學院 === 生物化學研究所 === 95 === Pigeon cytosolic NADP+-dependent malic enzyme ( EC 1. 1. 1. 40) catalyzes oxidative decarboxylation of malate to pyruvate and CO2 using NADP+ as an oxidant in the presence of divalent metal ion. Our previous studies indicated that N442A mutant showed dramatic decrease in catalytic activity (105-flod) compared to that of wild-type. In the crystal structure, N442 residue is located in the bottom of the active site distal to malate to directly involve in the catalysis. Therefore, N442 residue may have an important role in the structure of active site. Fluorescence quenching studies have been performed using charged KI as well as polar uncharged acrylamide quenchers. In both wild-type and N442A mutant, the Ksv values of both apoenzyme and enzyme-malate-Mg2+ tertiary complex for acrylamide are significantly larger than those of KI, indicating that most of tryptophan residues are not fully exposed to the solvent. Similar accessibility of tryptophan toward acrylamide was observed in the apo and tertiary compex of N442A mutant and tertiary complex of wild-type. Accessibility of tryptophans toward KI was greater in the tertiary complex than in the apoenzyme of both wild-type and N442A mutant. NADPH quenched the tryptophan fluorescence of both wild-type and N442A mutant. Since NADPH does not bind to tryptophan residue directly, the fluorescence quenching may result from conformational change upon addition NADPH. The Ksv values of enzyme-malate-Mg2+ tertiary complex of wild-type and N442A mutant for NADPH are 2- and 3-fold increase, respectively, compared to those of the apoenzyme. The fluorescence of 1 – anilinonaphalene (ANS), a hydrophobic-sensitive probe, is decreased about 40% in wild-type- malate-Mg2+ tertiary complex compared to that of wild-type apoprotein. N442A mutant showed about 70% decrease in tertiary complex. In conclusion, the structure of N442A mutant are different from the wild-type in both apoenzyme and enzyme-malate-Mg2+ complex. This structural difference may responsible for the dramatic decrease in the catalytic activity of N442A mutant.