Analysis of pigeon liver malic enzyme binding sites for NADP+ and L-malate by alanine scanning mutagenesis.
碩士 === 國防醫學院 === 生物化學研究所 === 84 === Cytosolic malic enzyme [(S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40] decarboxylates L- malate to yield pyruvate and CO2 with concomitant reduction of NADP+ to NADPH. This generated NA...
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ndltd-TW-084NDMC01070052015-10-13T14:34:57Z http://ndltd.ncl.edu.tw/handle/87930353408700566210 Analysis of pigeon liver malic enzyme binding sites for NADP+ and L-malate by alanine scanning mutagenesis. 利用定位突變法探討菸鹼醯胺腺▇呤二核▇酸磷酸及蘋果酸與胞漿蘋果酸▇結合之研究 Cherng-Chin Kuo 郭呈欽 碩士 國防醫學院 生物化學研究所 84 Cytosolic malic enzyme [(S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40] decarboxylates L- malate to yield pyruvate and CO2 with concomitant reduction of NADP+ to NADPH. This generated NADPH is a source of the reducing equivalents necessary for the de novo biosynthesis of long-chain fatty acid. The pigeon liver cytosolic malic enzyme is a tetramer composed of four identical subunits with molecular weight about 65,000 dalton. Based on the chemical modification studies, histidine and arginine residues were proposed to involve in the NADP+ binding and L-malate, respectively. Aligning of amino acid sequence of pigeon liver cytosolic malic enzyme with those from other species, twenty-two arginine and six histidine residues were located in the highly conserved regions. These highly conserved amino acid residues were mutated by the alanine scanning mutagenesis. These mutants were the subjects for preliminary kinetic studies. The apparent Km values for L-malate for R24A and R70A is increased by about 5-fold and 10-fold, respectively. Both the apparent Km values for Mn2+ for R24A and R70A are increased by about 10-fold. The kcat values for R24A and R70A are decreased by 10-fold and 1.5-fold, respectively. The kinetic parameters of all other mutants are similar to those for wild type. These results indicate that both of R24 and R70 may be involve in L-malate binding. The KmNADP+ values of all the mutants are almost identical to that of the wild type. It suggested that these amino acid residues may not involve in the nucleotide binding. Not all mutants showed as tetramer on native PAGE gel. All the mutants showed one protein band in molecular weight about 65 ,000 dalton on SDS PAGE gel. These results appear to indicat that these amino acid residues may participate in subunit''s interaction. Wei-Yuan Chou 周慰遠 1996 學位論文 ; thesis 62 zh-TW |
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碩士 === 國防醫學院 === 生物化學研究所 === 84 === Cytosolic malic enzyme [(S)-malate:NADP+ oxidoreductase
(oxaloacetate-decarboxylating), EC 1.1.1.40] decarboxylates L-
malate to yield pyruvate and CO2 with concomitant reduction of
NADP+ to NADPH. This generated NADPH is a source of the
reducing equivalents necessary for the de novo biosynthesis
of long-chain fatty acid. The pigeon liver cytosolic malic
enzyme is a tetramer composed of four identical subunits with
molecular weight about 65,000 dalton. Based on the chemical
modification studies, histidine and arginine residues were
proposed to involve in the NADP+ binding and L-malate,
respectively. Aligning of amino acid sequence of pigeon liver
cytosolic malic enzyme with those from other species,
twenty-two arginine and six histidine residues were located
in the highly conserved regions. These highly conserved amino
acid residues were mutated by the alanine scanning
mutagenesis. These mutants were the subjects for preliminary
kinetic studies. The apparent Km values for L-malate for R24A
and R70A is increased by about 5-fold and 10-fold,
respectively. Both the apparent Km values for Mn2+ for R24A
and R70A are increased by about 10-fold. The kcat values for
R24A and R70A are decreased by 10-fold and 1.5-fold,
respectively. The kinetic parameters of all other mutants are
similar to those for wild type. These results indicate that
both of R24 and R70 may be involve in L-malate binding. The
KmNADP+ values of all the mutants are almost identical to
that of the wild type. It suggested that these amino acid
residues may not involve in the nucleotide binding. Not all
mutants showed as tetramer on native PAGE gel. All the
mutants showed one protein band in molecular weight about 65
,000 dalton on SDS PAGE gel. These results appear to indicat
that these amino acid residues may participate in subunit''s
interaction.
|
author2 |
Wei-Yuan Chou |
author_facet |
Wei-Yuan Chou Cherng-Chin Kuo 郭呈欽 |
author |
Cherng-Chin Kuo 郭呈欽 |
spellingShingle |
Cherng-Chin Kuo 郭呈欽 Analysis of pigeon liver malic enzyme binding sites for NADP+ and L-malate by alanine scanning mutagenesis. |
author_sort |
Cherng-Chin Kuo |
title |
Analysis of pigeon liver malic enzyme binding sites for NADP+ and L-malate by alanine scanning mutagenesis. |
title_short |
Analysis of pigeon liver malic enzyme binding sites for NADP+ and L-malate by alanine scanning mutagenesis. |
title_full |
Analysis of pigeon liver malic enzyme binding sites for NADP+ and L-malate by alanine scanning mutagenesis. |
title_fullStr |
Analysis of pigeon liver malic enzyme binding sites for NADP+ and L-malate by alanine scanning mutagenesis. |
title_full_unstemmed |
Analysis of pigeon liver malic enzyme binding sites for NADP+ and L-malate by alanine scanning mutagenesis. |
title_sort |
analysis of pigeon liver malic enzyme binding sites for nadp+ and l-malate by alanine scanning mutagenesis. |
publishDate |
1996 |
url |
http://ndltd.ncl.edu.tw/handle/87930353408700566210 |
work_keys_str_mv |
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