α-Isopropylmalate synthase (α-IPMS) catalyses the first committed step in leucine biosynthesis in bacteria, including Neisseria meningitidis and Mycobacterium tuberculosis. It catalyses the condensation of α-ketoisovalerate (α-KIV) and acetyl coenzyme A (AcCoA) to form α-isopropylmalate (α-IPM). Like many key enzymes in biosynthesis, α-IPMS is inhibited by the end-product of the biosynthetic pathway, in this case leucine. α-IPMS is homodimeric, with monomers consisting of a (β/α)8-barrel catalytic domain, two subdomains and a C-terminal regulatory domain, responsible for binding leucine and providing feedback inhibition for leucine biosynthesis.
The exact mechanism of feedback inhibition in this enzyme is unknown, despite the elucidation of crystal structures with and without leucine bound. This thesis explores the nature of allosteric regulation in α-IPMS, including the effects of the regulatory domain and the importance of structural asymmetry on catalytic activity.
Chapter 2 details the characterisation of wild-type α-IPMS from N. meningitidis (NmeIPMS). This protein was successfully cloned, expressed and purified by metal-affinity and size-exclusion chromatography. NmeIPMS has similar characteristics to previously characterised α-IPMSs, being a dimer and demonstrating substrate binding affinities in the micromolar range. This enzyme has a turnover number of 13s⁻¹ and is sensitive to mixed, non-competitive inhibition by the amino acid leucine. Small angle X-ray scattering experiments reveal that the solution-phase structure of this protein is likely similar to existing crystal structures of other α-IPMSs.
In Chapter 3, substitutions of residues potentially involved in the binding and transmission of the leucine regulatory mechanism are described. Most of these amino acid substituted variants reduce enzyme sensitivity to leucine, and one variant is almost entirely insensitive to this inhibitor. Another of these variants demonstrates an unexpected decrease in substrate affinity, despite the substituted residue being located far from the active site.
The independence of α-IPMS domains is investigated in Chapter 4. The catalytic domains were isolated from NmeIPMS and the α-IPMS from M. tuberculosis (MtuIPMS), and found to be unable to catalyse the condensation of substrates, despite maintaining the wild-type structural fold. Complementation studies with Escherichia coli cells lacking the gene for α-IPMS show that the truncated variants are unable to rescue growth in these cells. Binding of α-KIV in the truncated NmeIPMS variant is much stronger than in the wild-type, and this may be the reason for lack of competent catalysis. A crystal structure was solved for the truncated variant of NmeIPMS and indicates that the regulatory domain is required for proper positioning of large regions of the protein. Two isolated regulatory domains from NmeIPMS were cloned, but with limited success in characterisation.
Finally, Chapter 5 describes substitutions made in MtuIPMS to affect relative domain orientations within the protein. Dimer asymmetry is investigated by substituting residues at the domain interfaces. These substitutions did have some effect on catalysis and inhibition, but did not show any change in average solution-phase structure.
These results are drawn together in the greater context of allostery in general in Chapter 6, along with ideas for future research in this field. This chapter reviews the insights gained into protein structure from this thesis, particularly the importance of residues at protein domain interfaces. The asymmetry in the α-IPMS structure is discussed, along with small-molecule binding regulatory domains.
| Identifer | oai:union.ndltd.org:canterbury.ac.nz/oai:ir.canterbury.ac.nz:10092/7599 |
| Date | January 2012 |
| Creators | Huisman, Frances Helen Adam |
| Publisher | University of Canterbury. Chemistry Department |
| Source Sets | University of Canterbury |
| Language | English |
| Detected Language | English |
| Type | Electronic thesis or dissertation, Text |
| Rights | Copyright Frances Helen Adam Huisman, http://library.canterbury.ac.nz/thesis/etheses_copyright.shtml |
| Relation | NZCU |
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