ENZYME FLEXIBILITY - A NEW CONCEPT IN RECOGNITION OF HYDROPHOBIC SUBSTRATES

The mechanism of recognition of hydrophobic substrates was investigate d using Escherichia coli aspartate aminotransferase (AspAT), E. coli a romatic amino acid aminotransferase (AroAT), and their chimeric enzyme (DY18), Surprisingly, broad substrate specificity was observed in the reaction of aminotransferases with hydrophobic substrates, The cataly tic efficiency increased with an increase in the side chain length of straight or branched-terminal aliphatic substrates, The straight-chain substrates catalysed with maximal efficiency were the 7-carbon substr ate in the case of AspAT and the 8-carbon substrate for AroAT and DY18 , Consecutive addition of single methylene groups to the substrate had a constant effect on the stabilization energy of the transition state relative to the unbound state, The dependency of binding energy on ea ch methylene group is usually interpreted as indicating hydrophobicity of the active site, However, we observed that AroAT and DY18 had diff erent dependencies although both enzymes have the same residues in the substrate-binding pocket, For substrates with more than 7 carbons, th e aminotransferases did not strictly distinguish between substrates wi th straight and branched side chains, These results suggest that the r ecognition of manifold hydrophobic substrates of different shapes migh t require not only the hydrophobicity of the active site but also enzy me flexibility.