Pyridoxal 5'-phosphate and analogs as probes of coenzyme-protein interaction in Baccillus alvei tryptophanase.
Trytophanase from Bacillus alvei was resolved from its coenzyme, pyridoxal phosphate, by treatment with cysteine followed by column chromatography. Spectrophotometric titration of apoenzyme with pyridoxal-P showed 1 mol of pyridoxal-P bound per 52,000 g of enzyme. Kinetic analysis of coenzyme binding showed hyperbolic activation curves with a Km of 1.6 muM. Pyridoxal-P was used as a natural active site probe in spectrophotometric studies to distinguish differences in the active center of holotryptophanase and reconstituted enzyme that were not apparent by other techniques. The pKa for holotryptophanase is 7.9 while the pKa for reconstituted apoenzyme is 8.4. Apotryptophanase binds 2-nor, 2'-methyl, 2'-hydroxy, 6-methyl, and N-oxide pyridoxal-P to form analog enzymes distinguishable on the basis of absorption spectra and relative activity in catalyzing both the alpha, beta-elimination and beta-replacement reactions of tryptophanase. Apoenzyme also binds pyridoxal but pyridoxal analog enzyme is not active.
D001051:Apoenzymes / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012695:L-Serine Dehydratase / Q000378:metabolism; D008190:Lyases / Q000378:metabolism*; D011485:Protein Binding; D011730:Pyridoxal / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014367:Tryptophan Synthase / Q000378:metabolism; D014368:Tryptophanase / Q000378:metabolism*
D001051:Apoenzymes; D003067:Coenzymes; D011730:Pyridoxal; D011732:Pyridoxal Phosphate; D008190:Lyases; D014368:Tryptophanase; D014367:Tryptophan Synthase; D012695:L-Serine Dehydratase