Active site model for gamma-aminobutyrate aminotransferase explains substrate specificity and inhibitor reactivities

A homology model for the pig isozyme of the pyridoxal phosphate-dependent enzyme gamma-aminobutyrate (GABA) aminotransferase has been built based mainly on the structure of dialkylglycine decarboxylase and on a multiple sequence alignment of 28 evolutionarily related enzymes. The proposed active site structure is presented and analyzed. Hypothetical structures for external aldimine intermediates explain several characteristics of the enzyme. In the GABA external aldimine model, the pro-S proton at C4 of GABA, which abstracted in the 1,3-azaallylic rearrangement interconverting the aldimine and ketimine intermediates, is oriented perpendicular to the plane of the pyridoxal phosphate ring. Lys 329 is in close proximity and is probably the general base catalyst for the proton transfer reaction. The carboxylate group of GABA interacts with Arg 192 and Lys 203, which determine the specificity of the enzyme for monocarboxylic omega-amino acids such as GABA. In the proposed structure for the L-glutamate external aldimine, the alpha-carboxylate interacts with Arg 445. Glu 265 is proposed to interact with this same arginine in the GABA external aldimine, enabling the enzyme to act on omega-amino acids in one half-reaction and on alpha-amino acids in the other. The reactivities of inhibitors are well explained by the proposed active site structure. The R and S isomers of beta-substituted phenyl and p-chlorophenyl GABA would bind in very different modes due to differential steric interactions, with the reactive S isomer leaving the orientation of the GABA moiety relatively unperturbed compared to that of the natural substrate. In our model, only the reactive S isomer of the mechanism-based inhibitor vinyl-GABA, an effective anti-epileptic drug known clinically as Vigabatrin, would orient the scissile C4-H bond perpendicular to the coenzyme ring plane and present the proton to Lys 329, the proposed general base catalyst of the reaction. The R isomer would direct the vinyl group toward Lys 329 and the C4-H bond toward Arg 445. The active site model presented provides a basis for site-directed mutagenesis and drug design experiments.