Kinetic and equilibrium studies on the activation of Escherichia coli K12 tryptophanase by pyridoxal 5'-phosphate and monovalent cations.
An improved purification of Escherichia coli K12 tryptophanase is presented. It is shown that the apoenzyme crystals, oxidized by exposure to air, can be reactivated by treatment with a reducing agent. The titration of sulfhydryl groups shows that four --SH groups are exposed and two are masked per protomer. The influence of two effectors, monovalent cations and the coenzyme pyridoxal 5'-phosphate, on the reactivity of --SH groups and the enzymatic activity was investigated. The --SH groups react more slowly in holo- than in apoenzyme in the presence of potassium ions. If these ions are replaced by sodium ions, the reactivity becomes the same. Potassium and ammonium ions, both activators, give sigmoidal activation curves. The sodium ion is a Michaelian inhibitor of potassium activation. The binding of pyridoxal 5'-phosphate was examined by kinetics and at equilibrium. The kinetics are shown to be very slow; the rate constants of the forward and reverse reactions have been measured. The binding equilibrium, examined with 3H-labeled pyridoxal 5'-phosphate, gives one site per protomer with a K-D value of (3.2 plus or minus 0.8) times 10-7 M. The K-m for pyridoxal-P was determined by activity measurements. The binding equilibrium is attained after several hours, giving a value of 4.2 times 10-7 M, being nearly identical with the dissociation constant and 5 times smaller than previously reported.
D001051:Apoenzymes / Q000378:metabolism; D002414:Cations, Monovalent / Q000494:pharmacology*; D003956:Dialysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014368:Tryptophanase / Q000302:isolation & purification / Q000378:metabolism*
D001051:Apoenzymes; D002414:Cations, Monovalent; D000644:Quaternary Ammonium Compounds; D013438:Sulfhydryl Compounds; D011732:Pyridoxal Phosphate; D012964:Sodium; D008190:Lyases; D014368:Tryptophanase; D011188:Potassium