Protein Backbone Engineering through Total Chemical Synthesis: New Insight into the Mechanism of HIV-1 Protease Catalysis

Total chemical synthesis by convergent chemical ligation was used to prepare a ‘backbone engineered’ 202-residue covalent dimer asymmetric form of the HIV-1 protease molecule. The Gly49-Ile50 peptide bond backbone –N(H)– atom, critically involved in H-bonding to substrates, was specifically replaced by an –O– atom in one flap only. The resulting enzyme analogue retained full intrinsic activity, demonstrating that enzyme–substrate hydrogen bonding at the Gly49-Ile50 peptide bond in only a single flap is sufficient for normal catalytic function.