Biochemical characterization and kinetic properties of alanine aminotransferase homologues partially purified from wheat (Triticum aestivum L.) |
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Affiliation: | 1. Neuroendocrine Unit, Massachusetts General Hospital, Boston, MA 02114, United States;2. Division of Cardiology, Massachusetts General Hospital, Boston, MA 02114, United States;3. Harvard Medical School, Boston, MA 02115, United States;1. Pediatric Department, Huntsville Hospital, Huntsville, AL;2. School of Medicine, University of Alabama, Birmingham, Huntsville, AL;1. Department of Internal Medicine and Clinical Nutrition, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Department of Endocrinology, Sahlgrenska University Hospital, Gröna Stråket 8, SE-413 45 Gothenburg, Sweden;2. Department of Neurosurgery, Sahlgrenska University Hospital, Göteborg, Sweden |
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Abstract: | Four homologues of alanine aminotransferase have been isolated from shoots of wheat seedlings and purified by saline precipitation, gel filtration, preparative electrophoresis and anion exchange chromatography on Protein-Pak Q 8HR column attached to HPLC. Alanine aminotransferase 1 (AlaAT1) and 2 (AlaAT2) were purified 303- and 452-fold, respectively, whereas l-glutamate: glyoxylate aminotransferase 1 (GGAT1) and 2 (GGAT2) were purified 485- and 440-fold, respectively. Consistent inhibition of AlaAT (EC 2.6.1.2) and GGAT (EC 2.6.1.4) activities by p-hydroxymercuribenzoate points on participation of cysteine residues in the enzyme activity. The molecular weight of AlaAT1 and AlaAT2 was estimated to be 65 kDa and both of them are monomers in native state. Nonsignificant differences between Km using alanine as substrate and catalytic efficiency (kcat/Km) for l-alanine in reaction with 2-oxoglutarate indicate comparable kinetic constants for AlaAT1 and AlaAT2. Similar kinetic constants for l-alanine in reaction with 2-oxoglutarate and for l-glutamate in reaction with pyruvate for all four homologues suggest equally efficient reaction in both forward and reverse directions. GGAT1 and GGAT2 were able to catalyze transamination between l-glutamate and glyoxylate, l-alanine and glyoxylate and reverse reactions between glycine and 2-oxoglutarate or pyruvate. Both GGATs also consisted of a single subunit with molecular weight of about 50 kDa. The estimated Km for GGAT1 (3.22 M) and GGAT2 (1.27 M) using l-glutamate as substrate was lower in transamination with glyoxylate than with pyruvate (9.52 and 9.09 mM, respectively). Moreover, distinctively higher values of catalytic efficiency for l-glutamate in reaction with glyoxylate than for l-glutamate in reaction with pyruvate confirm involvement of these homologues into photorespiratory metabolism. |
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