Determinants of Substrate Specificity in ω-Aminotransferases

Abstract

Ornithine aminotransferase and 4-aminobutyrate aminotransferase are related pyridoxal phosphate-dependent enzymes having different substrate specificities. The atomic structures of these enzymes have shown (i) that active site differences are limited to the steric positions occupied by two tyrosine residues in ornithine aminotransferase and (ii) that, uniquely among related, structurally characterized aminotransferases, the conserved arginine that binds the ␣-carboxylate of ␣-amino acids interacts tightly with a glutamate residue. To determine the contribution of these residues to the specificities of the enzymes, we analyzed site-directed mutants of ornithine aminotransferase by rapid reaction kinetics, x-ray crystallography, and 13 C NMR spectroscopy. Mutation of one tyrosine (Tyr-85) to isoleucine, as found in aminobutyrate aminotransferase, decreased the rate of the reaction of the enzyme with ornithine 1000-fold and increased that with 4-aminobutyrate 16-fold, indicating that Tyr-85 is a major determinant of specificity toward ornithine. Unexpectedly, the limiting rate of the second half of the reaction, conversion of ketoglutarate to glutamate, was greatly increased, although the kinetics of the reverse reaction were unaffected. A mutant in which the glutamate (Glu-235) that interacts with the conserved arginine was replaced by alanine retained its regiospecificity for the ␦-amino group of ornithine, but the glutamate reaction was enhanced 650-fold, whereas only a 5-fold enhancement of the ketoglutarate reaction rate resulted. A model is proposed in which conversion of the enzyme to its pyridoxamine phosphate form disrupts the internal glutamate-arginine interaction, thus enabling ketoglutarate but not glutamate to be a good substrate.Ornithine aminotransferase (Orn-AT) 3 and 4-aminobutyrate aminotransferase (GABA-AT) are members of a large family of pyridoxal 5Ј-phosphate (PLP)-dependent enzymes that catalyze a wide range of reactions on amino acids (1). Each enzyme operates by a mechanism, common to all aminotransferases ( Fig. 1), in which the cofactor shuttles between pyridoxaldimine and pyridoxamine forms by means of two coupled half-reactions (2, 3). The half-reaction converting ketoglutarate to glutamate is the same for both enzymes as well as for the majority of other aminotransferases. In this half-reaction, the chemical changes occur at the ␣-carbon. The substrate specificity of the enzymes thus arises from the other half-reaction that transfers an amino group distant from the ␣-carbon. For this reason, the enzymes are known as "-aminotransferases" (4). In the case of GABA-AT, the amino group transferred is the only amino group in the substrate molecule, whereas Orn-AT specifically selects the -amino group of ornithine despite the presence of a second amino group on C␣ with the same configuration as that in glutamate (3).The three-dimensional structures of both enzymes have been solved in the unliganded form as well as in complex with various substrate analogues (5-7). The enzymes have the same b...

Glutamine: precursor or nitrogen donor for citrulline synthesis?

“…In situ hybridization studies in neonatal small intestine have shown that the enzymes that metabolize citrulline into arginine are present only in the upper part of the villi, whereas the mRNA of the enzymes involved in the synthesis of citrulline were concentrated in the crypts (9). In adult rats, it has been shown inmunohistochemically that OAT is localized in villi but not in the crypts (28). This intraepithelial localization of enzyme activity adds an extra layer of complexity to the already complex interorgan trafficking of intermediates for citrulline synthesis.…”

Protein interactions and ligand binding: From protein subfamilies to functional specificity

“…2 and Movie S1). The SDPs include 3 positions that have been experimentally mutated, demonstrating their implication in determining substrate specificity (20). Despite not directly contacting the substrate, one of these positions has been experimentally shown to be one of the main determinants of substrate specificity (corresponding to residue 85 in PDB 1oat).…”

A subfamily of PLP-dependent enzymes specialized in handling terminal amines

“…In other words, in the transition between the first and second half-reactions of these α-KG-specific AT-II transaminases, the Glu residue acts as functional switch, triggered by the formation of PMP [21]. Such a switch allows these enzymes to be specific for aminogroup donors lacking an α-carboxylate while retaining the ability to bind the α-ketoacid acceptor 3 .…”

“…The presence of this salt bridge helps restrict the binding and reaction of undesired α-amino acids (e.g., glutamate) or α-keto acids, that are required only for the second part of the reaction [16,21,69].…”

“…Often the aminotransferases of the AT-II group are also referred to as ω-transaminases (ω-ATs; [17,18,[21][22][23]). Indeed the physiological reactions of these enzymes almost invariably involve a terminal amino group or at least (e.g., in the cases of ABS or FUMAT) an amino group not located in α-position with respect to a carboxylate (Table 1); compounds containing such groups can be broadly described as ω-amines [13] 1 .…”