Analysis of conformationally restricted alpha-ketoglutarate analogues as substrates of dehydrogenases and aminotransferases

Five synthetic, conformationally restricted alpha -ketoglutarate analogues were tested as substrates of a variety of dehydrogenases and aminotransfera ses. The compounds were found not to be detectable substrates of glutamate dehydrogenase, L-leucine dehydrogenase, L-phenylalanine dehydrogenase, lact ate dehydrogenase, malate dehydrogenase, glutamine transaminase K, aspartat e aminotransferase, alanine aminotransferase, and alpha -ketoglutarate dehy drogenase complex. However, two thermostable aminotransferases were identif ied that catalyze transamination between several L-amino acids (e.g., pheny lalanine, glutamate) and the alpha -ketoglutarate analogues of interest. Tr ansamination between L-glutamate (or L-phenylalanine) and the alpha -ketogl utarate analogues was found to be 0.13 to 1.08 mu mol/h/mg at 45 degreesC. The products resulting from transamination between L-phenylalanine and the alpha -ketoglutarate analogues were separated by reverse-phase HPLC, and th e newly formed amino acid analogues were analyzed by LC-MS in an ion select ive mode. In each case, the ions obtained were consistent with the expected product and a representative example is provided. The possibility existed that although the alpha -ketoglutarate analogues are not substrates of the dehydrogenases and most of the aminotransferases investigated, they might b e good inhibitors. Weak inhibition of aminotransferases and glutamate dehyd rogenase was found with some of the alpha -ketoglutarate analogues. The new ly available thermostable aminotransferases may have general utility in the synthesis of bulky L-amino acids from the corresponding alpha -keto acids. (C) 2001 Academic Press.