Kinetic and Equilibrium Studies on the Activation of Escherichia coli K12 Tryptophanase by Pyridoxal S-Phosphate and Monovalent Cations *
نویسنده
چکیده
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 end two are masked per protomer. The influence of two effecters, monovalent cations and the coenzyme pyridoxal S-phosphate, on the reactivity of -SH groups and the enzymatic activity was investigated. The -SH groups react more slowly in holothan 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 *Hlabeled pyridoxal 5’.phosphate, gives one site per protomer with a KD value of (3.2 i 0.8) x low7 M. The K,,, for pyridoxal-P was determined by activity measurements. The binding equilibrium is attained after several hours, giving a value of 4.2 x lo-’ IN, being nearly identical with the dissociation constant and 5 times smaller than previously reported.
منابع مشابه
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'-phosph...
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