The use of serine proteases as therapeutic agents may be greatly facilitated by an increasedunderstanding of how the structure of a protease dictates how it selects its substrate. The majority ofinvestigations of substrate recognition by serine proteases have primarily focused on substrate selectionand identification with respect to the protease's preferences for amino acids N-terminal to the scissilebond of the substrate, referred to as non-prime side selectivity. Little has been done in determining howthe amino acid(s) identities C-terminal to the scissile bond (prime side selectivity) influence(s) theprotease's selection of a substrate. The proposed experiments will explore and define this relationship.Using molecular modeling and mutagenesis techniques, the experiments are designed to evaluateprime-side selectivity by re-designing the amino acid architecture of the substrate binding sub-site of theprotein that associates with the amino acid immediately C-terminal to the scissile bond of the substrate.In addition to facilitating the development of proteolytic enzymes with desired specificity, the results fromthis study will also provide a mechanism to identify and establish general structural features of serineproteases that may govern selection of their natural substrate(s) based on the identities of the aminoacid constitution of the prime side binding sub-sites. The proposal is divided into three specific aims:
Specific Aim 1 is to construct S1' sub-site variants of the serine protease trypsin.
Specific Aim 2 is toconstruct a peptide library.
Specific Aim 3 is to kinetically and structurally characterize trypsin variantsthat exhibit altered selectivity.Relevance to Public Health: Re-engineered serine proteases such as tissue plasminogen activatorhave been demonstrated to be successful therapeutic agents in the treatment of stroke. An increasedunderstanding of how the architecture of the protease determines its activity may facilitate the reengineeringof other proteases for use as therapeutics in the treatment of other diseases or conditions.
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