Determinants of nucleotide-binding selectivity of malic enzyme.
Malic enzymes have high cofactor selectivity. An isoform-specific distribution of residues 314, 346, 347 and 362 implies that they may play key roles in determining the cofactor specificity. Currently, Glu314, Ser346, Lys347 and Lys362 in human c-NADP-ME were changed to the corresponding residues of...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2011-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3183043?pdf=render |
| _version_ | 1811339259820376064 |
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| author | Ju-Yi Hsieh Meng-Chun Chen Hui-Chih Hung |
| author_facet | Ju-Yi Hsieh Meng-Chun Chen Hui-Chih Hung |
| author_sort | Ju-Yi Hsieh |
| collection | DOAJ |
| description | Malic enzymes have high cofactor selectivity. An isoform-specific distribution of residues 314, 346, 347 and 362 implies that they may play key roles in determining the cofactor specificity. Currently, Glu314, Ser346, Lys347 and Lys362 in human c-NADP-ME were changed to the corresponding residues of human m-NAD(P)-ME (Glu, Lys, Tyr and Gln, respectively) or Ascaris suum m-NAD-ME (Ala, Ile, Asp and His, respectively). Kinetic data demonstrated that the S346K/K347Y/K362Q c-NADP-ME was transformed into a debilitated NAD⁺-utilizing enzyme, as shown by a severe decrease in catalytic efficiency using NADP⁺ as the cofactor without a significant increase in catalysis using NAD⁺ as the cofactor. However, the S346K/K347Y/K362H enzyme displayed an enhanced value for k(cat,NAD), suggesting that His at residue 362 may be more beneficial than Gln for NAD⁺ binding. Furthermore, the S346I/K347D/K362H mutant had a very large K(m,NADP) value compared to other mutants, suggesting that this mutant exclusively utilizes NAD⁺ as its cofactor. Since the S346K/K347Y/K362Q, S346K/K347Y/K362H and S346I/K347D/K362H c-NADP-ME mutants did not show significant reductions in their K(m,NAD) values, the E314A mutation was then introduced into these triple mutants. Comparison of the kinetic parameters of each triple-quadruple mutant pair (for example, S346K/K347Y/K362Q versus E314A/S346K/K347Y/K362Q) revealed that all of the K(m) values for NAD⁺ and NADP(+) of the quadruple mutants were significantly decreased, while either k(cat,NAD) or k(cat,NADP) was substantially increased. By adding the E314A mutation to these triple mutant enzymes, the E314A/S346K/K347Y/K362Q, E314A/S346K/K347Y/K362H and E314A/S346I/K347D/K362H c-NADP-ME variants are no longer debilitated but become mainly NAD⁺-utilizing enzymes by a considerable increase in catalysis using NAD⁺ as the cofactor. These results suggest that abolishing the repulsive effect of Glu314 in these quadruple mutants increases the binding affinity of NAD⁺. Here, we demonstrate that a series of E314A-containing c-NADP-ME quadruple mutants have been changed to NAD⁺-utilizing enzymes by abrogating NADP⁺ binding and increasing NAD⁺ binding. |
| first_indexed | 2024-04-13T18:22:38Z |
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| id | doaj.art-8e65d4efe04543e5977213774f6cc4cb |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-13T18:22:38Z |
| publishDate | 2011-01-01 |
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| spelling | doaj.art-8e65d4efe04543e5977213774f6cc4cb2022-12-22T02:35:21ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-0169e2531210.1371/journal.pone.0025312Determinants of nucleotide-binding selectivity of malic enzyme.Ju-Yi HsiehMeng-Chun ChenHui-Chih HungMalic enzymes have high cofactor selectivity. An isoform-specific distribution of residues 314, 346, 347 and 362 implies that they may play key roles in determining the cofactor specificity. Currently, Glu314, Ser346, Lys347 and Lys362 in human c-NADP-ME were changed to the corresponding residues of human m-NAD(P)-ME (Glu, Lys, Tyr and Gln, respectively) or Ascaris suum m-NAD-ME (Ala, Ile, Asp and His, respectively). Kinetic data demonstrated that the S346K/K347Y/K362Q c-NADP-ME was transformed into a debilitated NAD⁺-utilizing enzyme, as shown by a severe decrease in catalytic efficiency using NADP⁺ as the cofactor without a significant increase in catalysis using NAD⁺ as the cofactor. However, the S346K/K347Y/K362H enzyme displayed an enhanced value for k(cat,NAD), suggesting that His at residue 362 may be more beneficial than Gln for NAD⁺ binding. Furthermore, the S346I/K347D/K362H mutant had a very large K(m,NADP) value compared to other mutants, suggesting that this mutant exclusively utilizes NAD⁺ as its cofactor. Since the S346K/K347Y/K362Q, S346K/K347Y/K362H and S346I/K347D/K362H c-NADP-ME mutants did not show significant reductions in their K(m,NAD) values, the E314A mutation was then introduced into these triple mutants. Comparison of the kinetic parameters of each triple-quadruple mutant pair (for example, S346K/K347Y/K362Q versus E314A/S346K/K347Y/K362Q) revealed that all of the K(m) values for NAD⁺ and NADP(+) of the quadruple mutants were significantly decreased, while either k(cat,NAD) or k(cat,NADP) was substantially increased. By adding the E314A mutation to these triple mutant enzymes, the E314A/S346K/K347Y/K362Q, E314A/S346K/K347Y/K362H and E314A/S346I/K347D/K362H c-NADP-ME variants are no longer debilitated but become mainly NAD⁺-utilizing enzymes by a considerable increase in catalysis using NAD⁺ as the cofactor. These results suggest that abolishing the repulsive effect of Glu314 in these quadruple mutants increases the binding affinity of NAD⁺. Here, we demonstrate that a series of E314A-containing c-NADP-ME quadruple mutants have been changed to NAD⁺-utilizing enzymes by abrogating NADP⁺ binding and increasing NAD⁺ binding.http://europepmc.org/articles/PMC3183043?pdf=render |
| spellingShingle | Ju-Yi Hsieh Meng-Chun Chen Hui-Chih Hung Determinants of nucleotide-binding selectivity of malic enzyme. PLoS ONE |
| title | Determinants of nucleotide-binding selectivity of malic enzyme. |
| title_full | Determinants of nucleotide-binding selectivity of malic enzyme. |
| title_fullStr | Determinants of nucleotide-binding selectivity of malic enzyme. |
| title_full_unstemmed | Determinants of nucleotide-binding selectivity of malic enzyme. |
| title_short | Determinants of nucleotide-binding selectivity of malic enzyme. |
| title_sort | determinants of nucleotide binding selectivity of malic enzyme |
| url | http://europepmc.org/articles/PMC3183043?pdf=render |
| work_keys_str_mv | AT juyihsieh determinantsofnucleotidebindingselectivityofmalicenzyme AT mengchunchen determinantsofnucleotidebindingselectivityofmalicenzyme AT huichihhung determinantsofnucleotidebindingselectivityofmalicenzyme |