Determinants of substrate specificity in omega-aminotransferases.
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.
Markova
Michaela
M
School of Biosciences and School of Chemistry, Cardiff University, P. O. Box 911, CF23 6QU, Wales, United Kingdom.
Peneff
Caroline
C
Hewlins
Michael J E
MJ
Schirmer
Tilman
T
John
Robert A
RA
D000596:Amino Acids / Q000737:chemistry; D000613:Aminobutyrates / Q000737:chemistry; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods*; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D007532:Isoleucine / Q000737:chemistry; D007656:Ketoglutaric Acids / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009952:Ornithine / Q000737:chemistry; D009953:Ornithine-Oxo-Acid Transaminase / Q000737:chemistry; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013053:Spectrophotometry; D013379:Substrate Specificity; D013997:Time Factors; D000637:Transaminases / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
D000596:Amino Acids; D000613:Aminobutyrates; D007656:Ketoglutaric Acids; D011993:Recombinant Fusion Proteins; D007532:Isoleucine; D018698:Glutamic Acid; D014443:Tyrosine; D011732:Pyridoxal Phosphate; D004247:DNA; D003903:Deuterium; D009952:Ornithine; D000637:Transaminases; D009953:Ornithine-Oxo-Acid Transaminase