Using crystal structure data, site directed mutagenesis, and real- time kinetic assays, students designed, expressed, and purified engineered mutants of human insulin-degrading enzyme (IDE). Students designed their own IDE mutants following in silico analysis, used inverse PCR to generate mutant plasmid expression constructs, expressed mutant proteins in E. coli, and tested their function in vitro. Structural requirements for enzyme function were investigated. Students demonstrated a mastery of critical concepts in enzymology including principles of experimental design and evolutionary implications of enzyme structure−function relationships. Experimental methods for the analysis of macromolecular interactions and computational approaches to scientific inquiry were explored and applied. This investigation was conducted as part of the second-semester undergraduate biochemistry laboratory at Sacred Heart University.