Structural role of pyridoxal 5'-phosphate, pyridoxal 5'-phosphate analogs, and other agents in the association of subunits of Bacillus alvei apotryptophanase.
Bacillus alvei apotryptophanase readily dissociates at low protein concentration and sediments at 5.7 S (dimer) in 0.01 M potassium phosphate (pH 7.8) from 9 to 33 degrees. With temperature held constant at 9 degrees, increasing the potassium, sodium, or ammonium phosphate buffer concentration increases the sedimentation value to 8.0 S. Increasing the monovalent cation concentration alone does not have the effect. Imidazole and pyridoxal compete with phosphate, preventing the effect. Raising the temperature to 26 degrees in the presence of high concentrations of potassium phosphate increases the sedimentation constant to 9.4 S. The addition of pyridoxal-P converts the dimer to a 9.4S tetramer. The conversion is dependent upon coenzyme concentration, temperature, and the nature of monovalent cation present. The Km for pyridoxal-P for the sodium form of the enzyme is more than tenfold greater than the Km for the potassium form of the enzyme. 2'-Methyl, 2'-hydroxyl, 6-methyl, and the N-oxide of pyridoxal-P are active in the association of dimer to tetramer but to differing extents. Analogs altered in the 4'-formyl position are also inactive structurally. Anthranilic acid, a competitive inhibitor of tryptophan, and 8-anilino-1-naphthalenesulfonic acid (ANS), a competitive inhibitor of pyridoxal-P binding, are both active in affecting the dimer to tetramer association but tryptophan is not. The dimer and tetramer are spectrally distinguishable through circular dichroic measurements, fluroescence quenching with pyridoxal-P or pyridoxal, and fluorescence enhancement with ANS. Pyridoxal-P causes the release of ANS from an ANS-apoenzyme complex.
D001051:Apoenzymes; D001407:Bacillus / Q000201:enzymology*; D001667:Binding, Competitive; D002499:Centrifugation, Density Gradient; D002942:Circular Dichroism; D005470:Fluorometry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008190:Lyases; D046911:Macromolecular Substances; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature; D014368:Tryptophanase / Q000037:antagonists & inhibitors / Q000378:metabolism
D001051:Apoenzymes; D046911:Macromolecular Substances; D011732:Pyridoxal Phosphate; D008190:Lyases; D014368:Tryptophanase