Design of artificial enzymes by site directed mutagenesis and chemical engineering of a protein cavity

Semisynthetic enzymes can be created by covalent attachment of a catalytic active group to a protein scaffold. A well-characterized protein family known as fatty acid binding proteins (FABPs) consist of a large cavity for ligand binding. Intestinal fatty acid binding protein (IFABP) from rat is one FABP which has a cavity size of 600 Å3. By creating protein conjugates using IFABP, we have developed artificial enzymes that will perform different transformations in the interior of a protein by attached a metal containing or a non-metal containing functional group. Using the geometry of IFABP active site, the correct positioning of amino acid residues could provide high catalytic efficiency and selective recognition of substrates.; An artificial enzyme was assembled by conjugation of IFABP60 with a Manganese(III)Salen Complex via a disulfide bond to Cys 60 inside the cavity. The experimental results show that this conjugate exists in both dimeric and monomeric form and that it precipitates from solution. Gradual decomposition of the salen ligand was observed due to the space limitation inside the cavity. A helixless IFABP flavin conjugate, IFABP(Δ-15)45-FL which is able to catalyze the oxidation of dihydronicotinamides, has been synthesized. The kinetic studies illustrate that IFABP(Δ-15)45-FL can achieve the best catalytic rate acceleration using a dihydronicotinamide (NBuNH) as substrate. In comparison to flavopapain, IFABP(Δ-15)45-FL had larger K_m, however, the k_cat of IFABP(Δ-15)45-FL was 7–100-fold higher than that of the flavopapain.; The reactivity and selectivity of protein-pyridoxamine conjugates have been investigated by our group. In order to obtain artificial transaminases with higher catalytic efficiency and enantioselectivities, four double mutated IFABPs have been prepared and used for the synthesis of pyridoxamine (PX) or N-methylated pyridoxamine (MPX) conjugates. The rate accelerations of IFABP51K93A-MPX/PX and IFABP51K126-MPX/PX conjugates were found to be relatively low compared to the IFABP60-MPX/PX conjugates due to the precipitation of the mutant conjugates. Intriguingly, the IFABP38K93A-MPX showed a significantly high enantioselectivity and its PX conjugate had both the highest turnover and enantioselectivity at 25°C compared to other previously synthesized PX conjugates. The results described in this thesis demonstrated that the ‘chemogenetic’ approach provides the greatest chance for success in enzyme design.