Thermodynamics and Molecular Simulation Analysis of Hydrophobic Substrate Recognition by Aminotransferases

Abstract

Aromatic amino acid aminotransferase (AroAT) and aspartate aminotransferase (AspAT) are known as dualsubstrate enzymes, which can bind acidic and hydrophobic 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 analyses using substrates with different hydrophobicities were performed. They revealed that 1) amino acid substrate specificity (k max /K d ) depended on the affinity for the substrate (1/K d ) and 2) binding of the hydrophobic side chain was enthalpy-driven, suggesting that van der Waals interactions between the substrate-binding pocket and hydrophobic substrate predominated. Three-dimensional structures of AspAT and AroAT bound to ␣-aminoheptanoic acid were built using the homology modeling method. A molecular dynamic simulation study suggested that the outward-facing position of the Arg 292 side chain was the preferred state to a greater extent in AroAT than AspAT, which would make the hydrophobic 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 conformational rearrangement induced by substrate binding. These two mechanisms (positional preference of Arg and flexible conformation) may account for the high activity of AroAT toward hydrophobic substrates.Many enzymes show restricted specificities for single chemical types of substrate (1), but some have evolved binding pockets with dual specificities for different chemical groups (aminotransferases (Refs. 2 and 3) and cysteine protease cruzain (Ref. 4)). Escherichia coli aromatic amino acid aminotransferase (AroAT) 1 and aspartate aminotransferase (AspAT) are unique in being active toward two entirely different kinds of substrate (acidic and hydrophobic). These enzymes recognize a carboxyl group of an acidic substrate with side chain of arginine residue and recognize hydrophobic substrates in proportion to their hydrophobicities (3). These two kinds of substrates are accommodated in the one binding pocket of the enzyme.Structural determination and protein engineering techniques have made it relatively easy to define the key residues that recognize the polar and charged groups of a substrate. However, the mechanism responsible for hydrophobic substrate specificity is more complicated, because the complexity of interactions within the substrate binding pocket and multiple protein configurations must be taken into consideration. In many cases, a number of residues contribute to hydrophobic ligand specificity. There are also situations when the residues determining specificity do not interact directly with the substrate. Generally, hydrophobic side chains are well packed in the protein cores and a hydrophobic effect is one of the major factors involved in substrate recognition and protein folding (5). So far, three str...

Molecular Determinants for the Interaction of the Valvulopathic Anorexigen Norfenfluramine with the 5-HT2BReceptor

“…Table 1) of the V2.53L mutation on SNF binding was ϩ 1.66 kcal/mol. Others have measured the contribution of methyl group-methyl group interactions to ligand binding energy, with values ranging from ϩ0.4 kcal/mol to ϩ1.4 kcal/ mol (Bigler et al, 1993;Huang et al, 1995;Morton et al, 1995;Faergeman et al, 1996;Lu et al, 1997;Oue et al, 1997;Kawaguchi and Kuramitsu, 1998). The value calculated from our data (ϩ1.66 kcal/mol) is thus consistent with the loss of a 2.53 methyl group-SNF ␣-methyl group vdW interaction caused by the V2.53L mutation, a prediction of our modeling and MD simulations.…”

Analyses of microbial desulfurization reaction of alkylated dibenzothiophenes dissolved in oil phase

“…On the basis of these results, the hydrophobicity of the substrate due to the side chain was considered, which is often represented as the partition coefficient (log P) (Finizio et al, 1997). It has also been suggested that the hydrophobicity of the material correlates with biological activities such as: the substrate carbon number and the free energy difference in the reaction (Kawaguchi and Kuramitsu, 1998); the log P n-octanol/water value of the growth inhibitor and microbial activity determined on the basis of cell mass change (Yeom and Daugulis, 1999); and the log P n-octanol/water value and the in vitro apparent permeability (Kristl and Tukker, 1998). In this study, the mechanism of the oil/water two-phase reaction system with DBT monooxygenase activity as the initial reaction was examined using Mycobacterium G3, while considering the hydrophobicity of the substrate.…”

Free Energy Requirement for Domain Movement of an Enzyme

“…5, open circles) were studied with Cn substrates (12,13). When the free energy difference (⌬G T ‡ ) between the transition state (ES ‡ ) and the unbound enzyme plus the substrate (E ϩ S) (Fig.…”

“…3c). )With hydrophobic substrates, it is known that the catalytic efficiency increases in proportion to the side chain length of a series of straight aliphatic substrates (Cn substrates) (12,13). Surprisingly, consecutive additions of a single methylene group to the substrate (from C4 to C7) produce a constant effect on the stabilization energy of the transition state (ES ‡ ) relative to the unbound state (E ϩ S) (Refs.…”

“…E-PLP ϩ ␣-amino acid^E-PMP ϩ ␣-keto acid REACTION 1 AspAT is a unique "dual substrate" enzyme that catalyzes this reaction for both acidic and hydrophobic amino acids (12,13).…”