| Abstract: | Tryptophan synthase alpha 2 beta 2 complex containing 4'-3H]pyridoxal phosphate was reduced with sodium borohydride in the presence of various substrates and analogs in an attempt to trap reaction intermediates. Reduction in the presence of L-serine gave noncovalently bound radioactive material which was identified as phosphopyridoxylalanine, presumably resulting from reduction of the intermediate Schiff's base formed between pyridoxal phosphate and alpha-aminoacrylate. The tritium in this compound was located in the pro-R position at C-4', indicating that reduction of the Schiff's base double bond had occurred on the Si face at C-4'. On the other hand, analysis of phosphopyridoxyllysine obtained by hydrolysis of the reduced 3H]pyridoxal-P-alpha 2 beta 2 protein showed that the internal Schiff's base had been reduced on the C-4' Re face, suggesting a cofactor reorientation upon substrate binding. Analysis of phosphopyridoxylalanine from a reduction of unlabeled alpha 2 beta 2 complex in the presence of (2S,3R)-2,3-2H2]serine with tritiated sodium borohydride demonstrated the presence of tritium at C-4' (50%), C-2 (20%), and C-3 (30%). According to the configuration at C-3, reduction of the phosphopyridoxal-alpha-aminoacrylate Schiff's base has occurred from the same side of the molecule at C-4' and C-3. |
