THERMODYNAMICS AND MOLECULAR SIMULATION ANALYSIS OF HYDROPHOBIC SUBSTRATE RECOGNITION BY AMINOTRANSFERASES

Aromatic amino acid aminotransferase (AroAT) and aspartate aminotransf erase (AspAT) are known as dual-substrate enzymes, which can bind acid ic and hydro phobic substrates in the same pocket (Kawaguchi, S,, Nobe , Y., Yasuoka, J., Wakamiya, T,, Kusumoto, S,, and Kuramitsu, S, (1997 ) J, Biochem, (Tokyo) 122, 55-63), In order to elucidate the mechanism of hydrophobic substrate recognition, kinetic and thermodynamic analy ses using substrates with different hydrophobicities were performed. T hey revealed that 1) amino acid substrate specificity (k(max)/K-d) dep ended on the affinity for the substrate (1/K-d) and 2) binding of the hydrophobic side chain was enthalpy-driven, suggesting that van der Wa als interactions between the substrate-binding pocket and hydrophobic substrate predominated, Three-dimensional structures of AspAT and AroA T bound to alpha-aminoheptanoic acid were built using the homology mod eling method, A molecular dynamic simulation study suggested that the outward-facing position of the Arg(292) Side chain was the preferred s tate to a greater extent in AroAT than AspAT, which would make the hyd rophobic substrate bound state of the former more stable. Furthermore, AroAT appeared to have a more flexible conformation than AspAT. Such flexibility would be expected to reduce the energetic cost of conforma tional rearrangement induced by substrate binding. These two mechanism s (positional preference of Arg and flexible conformation) may account for the high activity of AroAT toward hydrophobic substrates.