Introduction and background
The enzyme BCAT (cytosolic isoform BCAT1 and mitochondrial isoform BCAT2) are catalyzing the conversion of branched chain amino acids isoleucine, leucine and valine to their corresponding keto acids for further degradation. The products of this process are used as sources of energy and building blocks for new molecules in cells. Interestingly, BCAT enzymes has recently been implicated in several forms of cancers, including the severe pancreatic cancer and glioblastoma (brain cancer), as these enzymes also support the rapid growing cancer cells in particular.
While the enzymatic mechanism of the BCAT enzymes is characterized at the structural and molecular level at large, we have data suggesting that the mechanism might be more complex and coordinated beyond what is the current state of knowledge. The two enzymes are dimeric, and with two active sites, there are several possibilities for coordination and regulation of the catalytic process in each site. The enzymes are highly reversible, also producing hydrophobic amino acids from keto acids under certain conditions. The activity is also regulated by red/oks sensitive elements.
Aim of project
The aim of this project is to use structural and biochemical methods to perform an in-depth investigation of the enzymatic mechanism. Different techniques and strategies will be used to understand the molecular details of the mechanism. Among the repertoire, we will design site-specific mutants with blocked active sites to understand the coordination between the two acive sites. We will use X-ray crystallography to study structures of captured reaction intermediates, and spectroscopy to follow the reaction in real-time under different conditions, such as presence of reducing agents, different ratios of substrates and products.
Project plan and methods
The project will consist of the following tasks and methods
- Use structural data to design site-specific mutants with partially blocked