The ubiquitin pathway regulates nearly all cellular processes in eukaryotes and consequently aberrations within the pathway can lead to a diversity of human diseases and disorders. Ubiquitin is a 76 residue protein that is conjugated to specific protein substrates via a cascade of enzymatic reactions that require an activating enzyme (E1), conjugating enzymes (E2s), and ligases (E3s). In humans there is a single E1, roughly 30 E2s, and hundreds of E3s. There are two main types of E3s that are structurally and functionally unrelated. RING E3s catalyze the direct transfer of ubiquitin from E2 to substrate while HECT E3s form a thioester intermediate with ubiquitin prior to transfer. In most cases E2s function with many E3s while E3s function with one or few homologous E2s. The study of diseases and disorders stemming from the ubiquitin pathway is complicated by the E1-E2-E3 hierarchy and by elusive E3 substrates. An attractive method to identify E3 substrates is to isolate specific E2-E3 interactions in the cell. One means to isolate E2-E3 interactions is to create altered specificity E2-E3 pairs that function with each other but not their wild type precursors nor other natural partners. The ability to create altered specificity E2-E3 pairs hinges on our understanding of the amino acid residues that dictate binding affinity and specificity. The intent of the work in this dissertation is to identify these determinants of binding affinity and specificity and rationally manipulate them. We have focused on the HECT class of E3s and began with the UbcH7-E6AP interaction as a model system. We used a combination of quantitative binding assays, mutagenesis, and ubiquitin transfer assays as well as rational and computation protein engineering to elucidate and manipulate the amino acid residues that govern E2-HECT binding affinity and specificity.