Biochemical investigation of the ubiquitin carboxyl-terminal hydrolase family

The proteasome is the machinery in eukaryotic cells that degrades protein and recycles the amino acids. Protein degradation is a highly regulated process which starts by the attachment of chains of ubiquitin, which serves as a tag that marks a protein for degradation. This function involves the work of several proteins at the proteasome that work either as ubiquitin chaperones, ubiquitin binders or cleave ubiquitin from the protein that is to be degraded. As this is a highly regulated process, various irregularities can have deleterious effects including the onset of disease, including cardiovascular, cancer, and neurological. The focus of this dissertation is to study how residues located within and outside the active site of Ubiquitin Carboxyl Terminal Hydrolase (UCH) deubiquinating enzymes (DUBS) help regulate these enzymes interaction with the ubiquitin. I will provide evidence that the putative oxyanion glutamine does function contribute to stabilization of the oxyanion intermediate. Secondly, I will show that evidence that glutamine may also serve another function within the active site by providing a CHO hydrogen bond that was previously thought not to exist within the active sites of cysteine proteases. Lastly, I will show that a conserved tryptophan in UCH37 has an effect on its catalytic viability