Coenzyme Site-directed Mutants of Photosynthetic A4-GAPDH Show Selectively Reduced NADPH-dependent Catalysis, Similar to Regulatory AB-GAPDH Inhibited by Oxidized Thioredoxin
| Autor: | Francesca Sparla, Paolo Pupillo, Mirko Zaffagnini, Alberto Ripamonti, Giuseppe Falini, Piera Sabatino, Simona Fermani, Paolo Trost |
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| Přispěvatelé: | SPARLA F, FERMANI S, FALINI G, ZAFFAGNINI M, RIPAMONTI A, SABATINO P, PUPILLO P., TROST P |
| Rok vydání: | 2004 |
| Předmět: |
Models
Molecular Chloroplasts Dehydrogenase Crystallography X-Ray Catalysis Cofactor Thioredoxins Spinacia oleracea Structural Biology Oxidoreductase Enzyme kinetics Photosynthesis Molecular Biology Glyceraldehyde 3-phosphate dehydrogenase chemistry.chemical_classification Binding Sites biology Enzyme structure Protein Structure Tertiary Kinetics Biochemistry chemistry Mutagenesis Site-Directed biology.protein NAD+ kinase Thioredoxin Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) Oxidation-Reduction NADP |
| Zdroj: | Journal of Molecular Biology. 340:1025-1037 |
| ISSN: | 0022-2836 |
| DOI: | 10.1016/j.jmb.2004.06.005 |
| Popis: | Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2′-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat (NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2′-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH. |
| Databáze: | OpenAIRE |
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