| It is widely accepted that much of the rate acceleration, in thiamin diphosphate (ThDP)-dependent enzymes, is provided by the cofactor. These enzymes are an incredibly successful and chemically diverse superfamily that has evolved to bind and maintain ThDP in the catalytically active V conformation. In addition to maintaining the V conformation, these enzymes further assist ThDP in catalysis by providing a substrate-docking site.;X-ray structures have revealed that the active sites of all ThDP enzymes contain a fulcrum residue thought responsible for maintaining the V conformation of ThDP. Additionally, these structures revealed that the active sites of ThDP enzymes exist at the interface between two monomers with each monomer contributing residues to the active site. Due to the manner in which the active sites of these enzymes are constructed, the minimal oligomerization state for activity is a dimer. As it happens, many of these enzymes exist as a tetramer for reasons not yet clear. Lastly, X-ray structures have also revealed very little conservation to active site residues, which would likely explain the diverse substrate specificity exhibited by members of the ThDP superfamily.;Using the tetrameric ThDP-dependent enzyme benzoylformate decarboxylase as a model system, saturation/site-directed mutagenesis, X-ray crystallography, and enzyme kinetics were used (i) to examine the long held tenet that a bulky hydrophobic residue provides a fulcrum by which the V conformation of the ThDP cofactor is maintained; (ii) to investigate the role of the tetramer in catalysis; and (iii) to determined how changes in the substrate-binding pocket affect specificity and activity. |
