New insights into the mechanism of dihydrodipicolinate synthase using isothermal titration calorimetry
| Autor: | Tatiana P. Soares da Costa, Andrew C. Muscroft-Taylor, Juliet A. Gerrard |
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| Rok vydání: | 2010 |
| Předmět: |
Models
Molecular Dihydrodipicolinate synthase Stereochemistry Allosteric regulation Protein Data Bank (RCSB PDB) Cooperativity Calorimetry Biochemistry Substrate Specificity Enzyme catalysis chemistry.chemical_compound Bacterial Proteins Biosynthesis Pyruvic Acid Escherichia coli Thermotoga maritima Protein Structure Quaternary Hydro-Lyases Schiff Bases Binding Sites Molecular Structure biology Isothermal titration calorimetry General Medicine Ligand (biochemistry) Protein Structure Tertiary chemistry biology.protein Thermodynamics Protein Binding |
| Zdroj: | Biochimie. 92:254-262 |
| ISSN: | 0300-9084 |
| Popis: | Thermodynamic binding information, obtained via isothermal titration calorimetry (ITC), provides new insights into the binding of substrates, and of allosteric inhibitor interactions of dihydrodipicolinate synthase (DHDPS) from Escherichia coli. DHDPS catalyses the first committed step in (S)-lysine biosynthesis: the Schiff-base mediated aldol condensation of pyruvate with (S)-aspartate semi-aldehyde. Binding studies indicate that pyruvate is a weak binder (0.023 mM) but that (S)-ASA does not interact with the enzyme in the absence of a Schiff-base with pyruvate. These results support the assignment of a ping pong catalytic mechanism in which enthalpically driven Schiff-base formation (DeltaH = -44.5 +/- 0.1 kJ mol(-1)) provides the thermodynamic impetus for pyruvate association. The second substrate, (S)-ASA, was observed to bind to a Schiff-base mimic (DeltaH = -2.8 +/- 0.1 kJ mol(-1)) formed through the reduction of the intermediate pyruvyl-Schiff-base complex. The binding interaction of (S)-lysine was characterised as a cooperative event in which an entropic pre-organisation step (TDeltaS = 17.6 +/- 1.1 kJ mol(-1)) precedes a secondary enthalpic association (DeltaH = -21.6 +/- 0.2 kJ mol(-1)). This allosteric association was determined to be of a mixed competitive nature in which heterotropic ligand cooperativity was observed to subtly influence the binding events. These results offer new insights into the inhibition of this enzyme, a validated antibiotic target. |
| Databáze: | OpenAIRE |
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