Enzyme flexibility: a new concept in recognition of hydrophobic substrates.

The mechanism of recognition of hydrophobic substrates was investigated using Escherichia coli aspartate aminotransferase (AspAT), E. coli aromatic amino acid aminotransferase (AroAT), and their chimeric enzyme (DY18). Surprisingly, broad substrate specificity was observed in the reaction of aminotransferases with hydrophobic substrates. The catalytic 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 substrate 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 each methylene group is usually interpreted as indicating hydrophobicity of the active site. However, we observed that AroAT and DY18 had different dependencies although both enzymes have the same residues in the substrate-binding pocket. For substrates with more than 7 carbons, the aminotransferases did not strictly distinguish between substrates with straight and branched side chains. These results suggest that the recognition of manifold hydrophobic substrates of different shapes might require not only the hydrophobicity of the active site but also enzyme flexibility.