title: The specificity and kinetic mechanism of branched-chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis.
abstract: Branched-chain amino acid aminotransferase (BCAT) plays a key role in the biosynthesis of hydrophobic amino acids (such as leucine, isoleucine and valine), and its substrate spectrum has not been fully explored or exploited owing to the inescapable restrictions of previous assays, which were mainly based on following the formation/consumption of the specific branched-chain substrates rather than the common amino group donor/acceptor. In our study, detailed measurements were made using a novel coupled assay, employing (R)-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans as an auxiliary enzyme, to provide accurate and reliable kinetic constants. We show that Escherichia coli BCAT can be used for asymmetric synthesis of a range of non-natural amino acids such as l-norleucine, l-norvaline and l-neopentylglycine and compare the kinetic results with the results of molecular modelling. A full two-substrate steady-state kinetic study for several substrates yields results consistent with a bi-bi ping-pong mechanism, and detailed analysis of the kinetic constants indicates that, for good 2-oxoacid substrates, release of 2-oxoglutarate is much slower than release of the product amino acid during the transamination reaction. The latter is in fact rate-limiting under conditions of substrate saturation.
  
  
  METHODS
  Branched-chain amino acid aminotransferase EC 2.6.1.42; (R)-2-hydroxyglutarate dehydrogenase EC 1.1.99.2.
authors.affiliation: School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Ireland.
authors.forename: Xuejing
authors.initials: X
authors.lastname: Yu
authors.forename: Xingguo
authors.initials: X
authors.lastname: Wang
authors.forename: Paul C
authors.initials: PC
authors.lastname: Engel
mesh_terms: D045850:Acidaminococcus / Q000201:enzymology*; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D055162:Biocatalysis; D004926:Escherichia coli / Q000201:enzymology*; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D008958:Models, Molecular; D009646:Norleucine / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity; D000637:Transaminases / Q000378:metabolism*; D014633:Valine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D007656:Ketoglutaric Acids; C034419:gamma-methylleucine; D009646:Norleucine; C005313:norvaline; D000429:Alcohol Oxidoreductases; C023858:2-hydroxyglutarate dehydrogenase; D000637:Transaminases; C019798:branched-chain-amino-acid transaminase; D007930:Leucine; D014633:Valine
keywords: branched-chain amino acid aminotransferase; kinetic mechanism; non-natural amino acids; rate-limiting step; substrate specificity