The role of R-281 and D-320 in the Reaction Mechanism of Human Dihydrolipoamide Dehydrogenase(E3)

• Main Authors: Shing-Ling Chang, 張杏菱
• Other Authors: Te-Chung Liu
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/11878860514026621608

碩士 === 中山醫學院 === 營養科學研究所 === 88 === Abstracrt by Shing-Ling Chang The mammalian α-ketoacid dehydrogenase complexes include pyruvate dehydrogenase (PDC) , α-ketoglutarate dehydrogenas(KGDC), and branched-chain α-ketoacid dehydrogenas complexes (BCKADC). Each enzyme complex is composed of three catalytic components,namely, α-ketoacid dehydrogenase (E1), dihydrolipoamide acyltransferase (E2), and E3.E1 and E2 show substrate specificity and E3 is the common component for all α-ketoacid dehydrogenase complexes. A deficiency in E3 leads to the deficiency of all these threeα-ketoacid dehydrogenase complexes. The symptoms of E3 deficiency include lactic acidosis, neurological dysfunction, increased serum amino acids and increased urinary organic acids. Since E3 plays an important role in the intermediary metabolism and in case of E3 deficiency, the patient will suffer serious metabolic disorder. The objective of this experiment is to study the reaction mechanism of dihydrolipoamide dehydrogenase(E3). Molecular sieving analysis showed that the R281N，R281K and D320N mutant protein was homodimer with the molecular-weight approximately to 102 kDa, as well as wild-type E3. The relative FAD content of these enzymes are E3：R281N：R281K：D320N＝100：96：100：83 . Kinetic analysis show the E3 and three mutant proteins, R281N、R281K and D320N, following two substrates ping-pong mechanism. The Kcat of these enzymes are E3：R281N：R281K：D320N=100：43：44：19.2 . Spectrophotometer analysis and midpoint reduction potential analysis show that mutant protein (R281N) of active disulfide midpoint reduction potential increased. The mutant protein (R281K) of FAD midpoint reduction potential increase and have not charge transfer. The mutant protein (D320N) reveals have not charge transfer and the FAD can not be reduced . In conclusion, the R281N and R281K may influence the charge transfer formation. The D320N may influence the ability of electrons transferring between FAD to NAD+.