Stability and Mechanism of Action of the Catalytic Subunit of Aspartate Transcarbamoylase

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When highly active catalytic (C) subunits isolated from Escherichia coli aspartate transcarbamoylase (ATCase) interact with regulatory subunits to form intact ATCase molecules, composed of 6 catalytic and 6 regulatory polypeptide chains, there are striking changes in the C subunits leading to a dramatic decrease in enzyme activity. Moreover, C subunits in the assembled complex undergo further extensive changes upon the addition of substrates, leading a more active enzyme. What are the structural and catalytic features which endow C trimers with these special properties thereby causing the C trimer to differ markedly from most oligomeric enzymes? Knowledge of the stability and possible fluctuations in structure would be invaluable. What is the role of the long helix connecting the N- terminal and C-terminal domains, each composed about 140 amino acids? What is the significance of shared active sites in the C trimer requiring joint participation of amino acid residues from adjacent chains? What role do interchain salt-links play in stabilizing C trimers and what is the role of distinct domains? What is the catalytic mechanism? Answering these questions requires many different mutant forms constructed by site-directed mutagenesis, determining the role of specific amino acids crucial for catalytic activity and correct folding of the chains, using NMR spectroscopy to measure pK's of residues implicated in catalysis, analyzing the pattern of activity over a broad pH range to evaluate acid-base catalysis, studying isotope effects to elucidate the catalytic mechanism, and studying binding of transition state analog inhibitors.