Fundamental studies of structure-function relationships of the FIV Protease (FIV PR) will be undertaken with the goal of increasing our understanding of this important class of retroviral enzyme. the human immunodeficiency virus-1 (HIV-1) PR is essential to the replication of infective virions, and is consequently the target of major drug design programs worldwide. Based on our own past work and the work of others there are already indications of unique structural and mechanistic features of HIV-1 PR compared with cell-encoded aspartyl proteinases that could serve as a basis for the design of a class of retroviral-selective inhibitors, as candidate therapeutics. Therefore we want to extend our studies to a more directly relevant model system, the FIV PR, and confirm the reality and generality of these features. Highly optimized total chemical synthesis will be used to prepare FIV PR for high resolution structural studies. The fundamental biochemical properties of the FIV PR will be determined, and routine assays will be developed. Substrate specificity will be compared to HIV-1 PR. Structure-function studies of FIV PR will be focused in two areas: 1. mechanism of the enzyme; and, 2. molecular origins of substrate specificity. These studies will take advantage of the unique ability of total chemical synthesis to introduce non-coded and geometrically- constrained structural features into the enzyme molecule. Analog PR molecules will be designed to test specific questions in each of these areas. Thus, the proposed role of Water301 in the catalytic mechanism will be evaluated by site-specific replacement of peptide bonds thought to be involved in H-bonding interactions with substrates/inhibitors through this water molecule. The ionization state of the catalytically active Asp side chains will be directly determined by single-atom labeling with 13C as an nmr reporter group. Rules governing substrate specificity will be deduced and tested by studies of FIV PR action on substrate mixtures, with mass spectrometric readout. The fundamental knowledge resulting from these studies of HIV PR will be important for the understanding of related clinically-relevant enzymes, such s the HIV-1 and other retroviral proteases, and cell-encoded aspartyl proteinases such as renin.
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