Studies on the determinants of cofactor specificity in pigeon liver malic enzyme
碩士 === 國防醫學院 === 生物化學研究所 === 92 === Malic enzymes use the dinucleotide cofactor, either NAD+ or NADP+, to catalyze the oxidative decarboxylation of malate to produce pyruvate and CO2 in the presence of divalent cations (Mg2+,Mn2+). Previous studies indicated that K340 was involved in the...
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ndltd-TW-092NDMC01070032016-06-17T04:16:18Z http://ndltd.ncl.edu.tw/handle/21725818599257468754 Studies on the determinants of cofactor specificity in pigeon liver malic enzyme 以定位突變探討鴿肝蘋果酸酶輔酶專一性之決定因素 Huang Chih-Heng 黃志恆 碩士 國防醫學院 生物化學研究所 92 Malic enzymes use the dinucleotide cofactor, either NAD+ or NADP+, to catalyze the oxidative decarboxylation of malate to produce pyruvate and CO2 in the presence of divalent cations (Mg2+,Mn2+). Previous studies indicated that K340 was involved in the binding of 2’-phosphate group of NADP+. However, its mutant showed little preference to NAD+. Therefore, it suggested that there are other amino acid residues are involved in the cofactor specificity. Base on the secondary structure alignment and the superimposition of the tertiary structure of the human mitochondrial NAD+- dependent malic enzyme(m-NAD-ME) and the pigeon cytosolic NADP+- dependent malic enzyme(c-NADP-ME), 15 mutaions in the region around the 2’-phosphate group of NADP+ cofactor have been introduced. Only 3 mutants shift the cofactor specificity. Compared with the wild type pigeon c-NADP- ME, the kinetic data showed that the Km NADP of S325K, K326D, K326Y, K340A and K340N were 6~75-fold higher and the Km NAD were 6~34-fold lower. The values of NADP-specificity (kcat/Km,,NADP/kcat/Km,,NAD) of the double mutant K326Y/K340D and the triple mutant S325K/K326D/ K340N are 0.05 and 0.15, respectively, which are close to the value of human m-NAD-ME(0.11). In conclusion, S325, K326 and K340 of pigeon c-NADP- ME are involved in the dinucleotide cofactor specificity. However, all these mutants demonstrated a dramatic decrease in their kcat values. It suggests that other regions of the protein, yet to be identified, might also be involved in the dertermination of cofactor specificity. 周慰遠 2004 學位論文 ; thesis 105 zh-TW |
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碩士 === 國防醫學院 === 生物化學研究所 === 92 === Malic enzymes use the dinucleotide cofactor, either NAD+ or NADP+, to catalyze the oxidative decarboxylation of malate to produce pyruvate and CO2 in the presence of divalent cations (Mg2+,Mn2+). Previous studies indicated that K340 was involved in the binding of 2’-phosphate group of NADP+. However, its mutant showed little preference to NAD+. Therefore, it suggested that there are other amino acid residues are involved in the cofactor specificity. Base on the secondary structure alignment and the superimposition of the tertiary structure of the human mitochondrial NAD+- dependent malic enzyme(m-NAD-ME) and the pigeon cytosolic NADP+- dependent malic enzyme(c-NADP-ME), 15 mutaions in the region around the 2’-phosphate group of NADP+ cofactor have been introduced. Only 3 mutants shift the cofactor specificity. Compared with the wild type pigeon c-NADP- ME, the kinetic data showed that the Km NADP of S325K, K326D, K326Y, K340A and K340N were 6~75-fold higher and the Km NAD were 6~34-fold lower. The values of NADP-specificity (kcat/Km,,NADP/kcat/Km,,NAD) of the double mutant K326Y/K340D and the triple mutant S325K/K326D/ K340N are 0.05 and 0.15, respectively, which are close to the value of human m-NAD-ME(0.11). In conclusion, S325, K326 and K340 of pigeon c-NADP- ME are involved in the dinucleotide cofactor specificity. However, all these mutants demonstrated a dramatic decrease in their kcat values. It suggests that other regions of the protein, yet to be identified, might also be involved in the dertermination of cofactor specificity.
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author2 |
周慰遠 |
author_facet |
周慰遠 Huang Chih-Heng 黃志恆 |
author |
Huang Chih-Heng 黃志恆 |
spellingShingle |
Huang Chih-Heng 黃志恆 Studies on the determinants of cofactor specificity in pigeon liver malic enzyme |
author_sort |
Huang Chih-Heng |
title |
Studies on the determinants of cofactor specificity in pigeon liver malic enzyme |
title_short |
Studies on the determinants of cofactor specificity in pigeon liver malic enzyme |
title_full |
Studies on the determinants of cofactor specificity in pigeon liver malic enzyme |
title_fullStr |
Studies on the determinants of cofactor specificity in pigeon liver malic enzyme |
title_full_unstemmed |
Studies on the determinants of cofactor specificity in pigeon liver malic enzyme |
title_sort |
studies on the determinants of cofactor specificity in pigeon liver malic enzyme |
publishDate |
2004 |
url |
http://ndltd.ncl.edu.tw/handle/21725818599257468754 |
work_keys_str_mv |
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