Structural and mechanistic studies of Ser/Lys dyad proteases.

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The goal of this research program is to gain insight into the structural and mechanistic details of architecturally unrelated proteolytic enzymes that have evolved to utilize the unusual Ser/Lys catalytic dyad mechanism for the hydrolysis of the peptide bond in their substrates. It is proposed that these enzymes provides a microenvironment for the epsilon-amino group of the catalytic lysine such that it is able to become deprotonated and thereby serve as the general-base by abstracting the proton from the catalytic serine hydroxyl, activating it for nucleophilic attack on the scissile carbonyl of the protein substrate. Ultimately we will use X-ray crystallography to create a molecular movie of these enzymes in action by trapping each stage of the reaction coordinate. Discovering the similarities and differences between the enzymes at each stage of their catalytic cycle and correlate the structural data with spectroscopic and kinetic data will allow us to understand the essential features of the Ser/Lys dyad mechanism and possibly provide new insights into enzyme catalyzed hydrolysis reactions in general. Many of the Ser/Lys proteases are essential enzymes in bacteria and viruses, therefore we will to use the information we glean from our investigations to develop inhibitors that work as anti-bacterial and anti-viral compounds. In the proposed funding period of this research program we are continuing our investigation into the molecular workings of VP4 proteases which functions to process the polyprotein in birnaviruses, a virus that infects fish and poultry and is the cause of diseases that result in significant lost revenues in the both the aquaculture and agriculture industries. We will also be investigating TraF protease, an enzyme involved in bacterial conjugative pili assembly and interestingly is proposed to have both protease and ligase activity to produce a cyclized protein product. Canada will benefit socially and financially from the commercialization of antibiotic and antiviral compounds that result for the investigation in these enzymes. Canada will also benefit from the training of highly qualifies personnel. Students will become skilled and knowledgeable in fields that are highly sought after in both academic and industrial sectors.