Probing a Functional Role of GLU141 in Porcine Cytosolic Aspartate Aminotransferase

Suginohara, K.; Hamasaki, N; Tanase, Sumio; Fukumoto, Y.; Morino, Yoshimasa

doi:10.1016/b978-0-08-040820-0.50040-6

Enzymes Dependent on Pyridoxal Phosphate and Other Carbonyl Compounds as Cofactors 1991

DOI: 10.1016/b978-0-08-040820-0.50040-6

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Probing a Functional Role of GLU141 in Porcine Cytosolic Aspartate Aminotransferase

K. Suginohara

N Hamasaki

Sumio Tanase

et al.

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1998

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“…The major difference is the presence of Glu 141 in AroAT and Pro at the same position in AspAT. Previous research showed that Glu 141 only has slight effect on the enzymatic activity of cAspAT (66). However, the importance of Glu 141 in AroAT is still unclear, because Glu 141 of AroAT can interact with Arg 292 outside the substrate-binding pocket, whereas that of cAspAT may be not be able to do so.…”

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Thermodynamics and Molecular Simulation Analysis of Hydrophobic Substrate Recognition by Aminotransferases

Kawaguchi¹,

Kuramitsu

1998

Journal of Biological Chemistry

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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...

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confidence: 90%

Thermodynamics and Molecular Simulation Analysis of Hydrophobic Substrate Recognition by Aminotransferases

Kawaguchi¹,

Kuramitsu

1998

Journal of Biological Chemistry

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Probing a Functional Role of GLU141 in Porcine Cytosolic Aspartate Aminotransferase

Cited by 1 publication

References 3 publications

Thermodynamics and Molecular Simulation Analysis of Hydrophobic Substrate Recognition by Aminotransferases

Thermodynamics and Molecular Simulation Analysis of Hydrophobic Substrate Recognition by Aminotransferases

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