Serah Kimani's poster title is a tongue twister: Unexpected reactions resulting from mutating catalytic residues in an amidase reveal the role of the catalytic unit. 'Unexpected' says it well, though.
A doctoral student based in UCT's Electron Microscope Unit and the Department of Molecular and Cell Biology, Kimani and unit director Professor Trevor Sewell were trying new approaches to work out the mechanisms of an enzyme known as amidase. While they are useful as biocatalysts in the synthesis of fine chemicals and pharmaceuticals, not much is understood about the catalytic mechanisms of amidases, explains Kimani, who graduates this week.
So they mutated each one of the amidase's active site residues, which are the amino acids directly responsible for the catalysis in the enzyme. Then they reacted the mutated enzyme with known substrates - molecules on which the enzyme acts - and, finally, looked at the results using mass spectroscopy and high-resolution crystallography.
Conventional wisdom, says Sewell, suggested that they would see only one derived biochemical compound, or covalent intermediate, known as a thioester. Instead they found two additional intermediates that do not occur in the natural enzyme. From these observations they then deduced the enzyme's mechanism.
As expected, their approach has raised some questions. Kimani and Sewell would also really like to observe how these unexpected intermediates are actually formed, but the difficulties they encountered with doing this experimentally have encouraged them to tackle the problem using theoretical quantum mechanical methods.
For now, though, the work has captured the attention of the biochemical world. Notably at the XXII Congress and General Assembly of the International Union of Crystallography in Madrid, Spain, in August, a large international conference where Kimani was named the winner of the Research Collaboratory for Structural Bioinformatics Protein Data Bank (or RCSB PDB) poster prize.
"I had to answer a lot of questions and do a lot of explaining," she recalls.
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