Structural and mechanistic studies of pyridoxal-5'-phosphate dependent enzymes.
A model for the active-site of pyridoxal-5'-phosphate dependent carboxylases has been developed based on the known structural and mechanistic features of these enzymes. Comparison of the known structural and mechanistic features of aspartate aminotransferase with those of the model for pyridoxal-5'-phosphate dependent decarboxylases indicated that it might be feasible to convert aspartate aminotransferase into a decarboxylase using site-directed mutagenesis techniques. The possibility was investigated using computer graphics and molecular modelling techniques. The various AAT mutant structures were created on the framework provided by the crystal structure of mAAT. The A257H R386Q mutant was modelled in detail. Multiple starting conformers were generated for the A257H R386Q mutant and these were energy minimized to allow the prediction of the structure and likely decarboxylase activity of the A257H R386Q mutant. The S257H R386l mutant of E.coli AAT was created. The mutant enzyme was tested under a variety of conditions and with various substrates but was found to be inactive as a decarboxylase and also inactive as a transaminase. The lack of activity is thought to be due to the loss of Arg 386 which plays a key role in a conformational change necessary for catalysis. Other possible mutations were also considered and modelled. The extent of -hydrogens observed when L-aspartic and L-glutamic acids are incubated in deuterium oxide with cytosolic aspartate aminotransferase is demonstrated to be non-stereoselective. Application of the model for pyridoxal-5'-phosphate dependent decarboxylases to the aligned sequences of mammalian, trypsanomal and yeast ornithine decarboxylase, an unusual pyridoxal-5'-phosphate dependent decarboxylase, permitted identification of the probable pyridoxal-5'-phosphate binding lysine residue.