The homotetrameric enzyme dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) from Escherichia coli was used as a model for probing oligomeric structure in enzymes. Dimeric mutants of this enzyme have been found in previous work to be largely inactive, due to the trapping of a covalent adduct. Partial restoration of catalytic activity has been achieved by incubation in the presence of the substrate pyruvate to displace the adduct. It was hypothesized that the buttressing of dimeric units against one another in the wildtype tetrameric form of DHDPS provides stability in the dimer interface, necessary to maintain optimum catalytic performance and substrate specificity. We hypothesized that buttressing a dimeric DHDPS mutant against a surface would result in restoration of catalytic activity by mimicking the buttressing proposed to occur in the tetrameric structure. To test this hypothesis, dimeric DHDPS mutants were immobilised against an agarose support and the immobilised enzymes characterised. Three DHDPS mutants were prepared, the double mutant DHDPS-C20S/L167C was produced by mutagenesis and a crystal structure obtained in collaboration with Dr Renwick Dobson. Two other mutants, DHDPS-Ll67C and DHDPS-Ll97Y were also over expressed and purified. The quaternary structures of the three mutants were characterised in solution, DHDPS-Ll67C was determined to be tetrameric, DHDPS-C20S-Ll67C was found to equilibrate between tetramer and dimer and DHDPS-Ll97Y was confirmed as a dimer, consistent with previous findings. Modification experiments indicated that the sulfhydryl groups of DHDPS-C20S/L167C were available for immobilisation. Activation experiments indicated that both DHDPS-Ll67C and DHDPS-Ll97Y activated. These results were in accord with those of others in indicating that the displacement of an a-ketoglutarate adduct from the active site was responsible for the activation of mutant DHDPS enzymes. Wild-type DHDPS and the mutants were immobilised through amine and sulfhydryl groups. The free and immobilised enzymes were rigorously characterised, with thermal stability, pH optima, kinetic and lysine inhibition properties determined and compared to wild-type DHDPS. Following immobilisation, substrate affinity was found to decrease for wild-type and mutant enzymes, wild-type KmPyr = 0.26 mM free, 0.8-1.2 mM immobilised, Km(S)-ASA = 0.10 mM free, 1.5-2.5 mM immobilised. Lysine inhibition was determined to be largely unaffected by immobilisation. The largest change in K, was an increase to double that of the free enzyme. Restoration of some catalytic activity was found following the immobilisation of dimeric DHDPS-Ll97Y, the immobilised enzyme was 31 ± 12% more active than free DHDPS-Ll97Y. DHDPS-C20S/L167C was also found to immobilise as a dimer. Comparison ofthe immobilised DHDPS-C20S/L167C dimer with a derivatised free dimeric form ofthis enzyme indicated that an increase from 3% to 9% of wild-type activity had resulted from immobilisation. These results supported the hypothesis that buttressing of a dimeric mutant of DHDPS against a support surface would increase catalytic activity and that buttressing across the dimerdimer interface is essential for optimal catalytic activity in DHDPS enzymes.
| Identifer | oai:union.ndltd.org:canterbury.ac.nz/oai:ir.canterbury.ac.nz:10092/1469 |
| Date | January 2007 |
| Creators | Baxter, Chris Logan |
| Publisher | University of Canterbury. Biological Sciences |
| Source Sets | University of Canterbury |
| Language | English |
| Detected Language | English |
| Type | Electronic thesis or dissertation, Text |
| Rights | Copyright Chris Logan Baxter, http://library.canterbury.ac.nz/thesis/etheses_copyright.shtml |
| Relation | NZCU |
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