Recent studies of rat branched-chain ct-ketoacid dehydrogenase (BCKDH) implicated several conserved residues surrounding the phosphorylation site 1 serine as active site residues with critical roles in oxidative decarboxylation. Mutation of arginme 288, a residue conserved in all a-ketoacid dehydrogenases, abolished enzyme activity but not the binding of thiamine pyrophosphate (TPP), suggesting a possible role for this residue in a-ketoacid binding. Mutation of histidine 292 to alanine abolished enzyme activity as well as TPP binding indicating that this residue, adjacent to the phosphorylation site 1 serine, is critical for (factor binding. We now show that mutation of the highly conserved residue glutamate 194 to alanine also abolished dehydrogenase activity as well as TPP binding. Previous modeling studies of other TPP-dependent enzymes implicated this conserved residue in binding the magnesium-chelated pyrophosphate moiety of TPP. From these data we propose an active site topology which brings the conserved TPPbinding domain into close proximity to the conserved residues surrounding the phosphorylation site 1 serine. Pyridoxal-5'-phosphate was found to inactivate BCKDH in a time dependent fashion, resulting in an observable peak of absorption at 328 nm. Spectral analysis of pyridoxylated E194A mutant BCKDH showed that this mutation did not affect the reaction with pyridoxal-5'-phosphate. This result implies that pyridoxal-5'-phosphate, which lacks the chelated pyrophosphate moiety of TPP, may bind to the TPP-binding site of wild-type and E194 mutant BCKDH. Alternatively, pyridoxal-S'-phosphate may be reacting not as a TPP analogue but as a probe for anion binding sites such as the a-ketoacid binding site. Further research will be designed to investigate this hypothesis.
|Original language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology