2BYJ

Ornithine aminotransferase mutant Y85I

Summary for 2BYJ

• Entry DOI: 10.2210/pdb2byj/pdb
• Related: 1GBN 1OAT 2CAN 2OAT
• Descriptor: ORNITHINE AMINOTRANSFERASE, PYRIDOXAL-5'-PHOSPHATE (3 entities in total)
• Functional Keywords: transferase, disease mutation, transit peptide, mitochondrion, plp-dependent enzyme, polymorphism, pyridoxal phosphate
• Biological source: HOMO SAPIENS (HUMAN)
• Cellular location: Mitochondrion matrix : P04181
• Total number of polymer chains: 3
• Total formula weight: 146372.38

Authors

Markova, M.,Peneff, C.,Hewlins, M.J.E.,Schirmer, T.,John, R.A. (deposition date: 2005-08-02, release date: 2005-09-07, Last modification date: 2023-12-13)

Primary citation

Markova, M.,Peneff, C.,Hewlins, M.J.E.,Schirmer, T.,John, R.A.
Determinants of Substrate Specificity in Omega-Aminotransferases.
J.Biol.Chem., 280:36409-, 2005

PubMed 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 alpha-carboxylate of alpha-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 13C 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 delta-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.

PubMed: 16096275
DOI: 10.1074/JBC.M506977200

Experimental method

X-RAY DIFFRACTION (3.02 Å)