Disruption of quaternary structure in Escherichia coli dihydrodipicolinate synthase (DHDPS) generates a functional monomer that is no longer inhibited by lysine
Autor: | Juliet A. Gerrard, F. Grant Pearce, Andrew C. Muscroft-Taylor, Renwick C. J. Dobson, Matthew A. Perugini, Ryan J. Catchpole |
---|---|
Rok vydání: | 2010 |
Předmět: |
Models
Molecular Dihydrodipicolinate synthase Stereochemistry Protein subunit Allosteric regulation Biophysics Biochemistry Catalysis Substrate Specificity Protein structure Enzyme Stability Point Mutation Enzyme kinetics Binding site Protein Structure Quaternary Molecular Biology Hydro-Lyases chemistry.chemical_classification Binding Sites biology Escherichia coli Proteins Lysine Kinetics Enzyme chemistry biology.protein Protein quaternary structure |
Zdroj: | Archives of biochemistry and biophysics. 503(2) |
ISSN: | 1096-0384 |
Popis: | Escherichia coli dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52), a natively homotetrameric enzyme was converted to a monomeric species through the introduction of destabilising interactions at two different subunit interfaces allowing exploration of the roles of the quaternary structure in affecting catalytic competency. The double mutant DHDPS-L197D/Y107W displays gel filtration characteristics consistent with a single non-interacting monomeric species, which was confirmed by sedimentary velocity experiments. This monomer was shown to be catalytically active, but with reduced catalytic efficiency ( k cat = 9.8 ± 0.5 s −1 ), displaying 8% of the specific activity of the wild-type enzyme. The Michaelis constants for the substrates pyruvate and for ( S )-aspartate semialdehyde increased by an order of magnitude, indicating that quaternary structure plays a significant role in substrate specificity. This monomeric species exhibited an enhanced propensity for aggregation and inactivation, indicating that whilst the oligomerization is not an intrinsic criterion for catalysis, higher oligomeric forms may benefit from both increased catalytic efficiency and diminished aggregation propensity. Furthermore, allosteric inhibition by ( S )-lysine was abolished for DHDPS-L197D/Y107W, confirming the importance of the dimeric unit as the minimal functional assembly for efficient ( S )-lysine binding. |
Databáze: | OpenAIRE |
Externí odkaz: |