The immediate goal of this grant is to examine the mechanism of ubiquitin- protein conjugation as part of a continuing interest in the multi-enzyme pathway of ATP, ubiquitin-dependent protein degradation. This system is implicated in a spectrum of fundamental regulatory processes within the cell for which an understanding of the mechanism and specificity of this novel post-translational modification is critical. The Specific Aims of the proposal are: (1)Characterize the E1 inhibitor of Manduca sexta intersegmental muscle. The low molecular weight (8 kDa) heat-stable E1 inhibitor proposed to regulate conjugation within ISM during eclosion will be purified and its mechanism of inhibition characterized. The putative high molecular weight antagonist of the inhibitor will also be examined. (2)Map the function of specific ubiquitin amino acid residues. Kinetic, thermodynamic, and site-directed mutagenesis will be exploited to map the function(s) of residues on ubiquitin with respect to contact surfaces for binding enzymes of the pathway, the contribution of such binding energy to the catalytic efficiency of E1, and the structural correlates on ubiquitin that define linkage specificity in E3-independent multi-ubiquitination. (3)Examine the mechanism of E214K. Kinetic and thermodynamic studies will be used to examine the relative affinities of E2 and its ubiquitin thiolester to activating enzyme and isopeptide ligase to test hypotheses on the competition of different isozymes for binding to these proteins. Site- directed mutagenesis will be used to elucidate the mechanism of transthiolation and conjugate formation. (4)Develop E2 affinity methods for isolating E3 isozymes. Affinity methods will be developed for using E2-linked and E2-ubiquitin thiolester analog- linked affinity columns to isolate putative E2-specific E3 isozymes.