ON THE ROLE OF PYRIDOXAL PHOSPHATE FOR AMP-DEPENDENT THREONINE DEAMINASE OF ESCHERICHIA COLI

Abstract

Threonine deaminase of anaerobically grown Es coli was purified about 125-fold by sonic disintegration, streptomycin treatment, [NH4]2 SO4 fractionation, Sephadex G-200 gel fractionation and DEAE-Sephadex column chromatography. Inclusion of AMP and [beta]-mercaptoethanol at 5 m[image] each throughout the purification procedures made the above degree of purification with 30% yield possible by protecting the enzyme against inactivation. The purified enzyme exhibited an absorption maximum at 404m[mu] and a fluorescence spectrum with a maximum emission at 510 m[mu] upon activation at 420 m[mu]. Resolution of pyridoxal phosphate from the threonine deaminase was performed by hydroxylamine treatment followed by gel filtration. Pyridoxal phosphate was the most effective reactivator and pyridoxamine phosphate was slightly effective for the apoenzyme. Pyridoxal, pyridoxamine and pyridoxine were without effect. The Km for pyridoxal phosphate was 7.7 [mu][image], and the presence of AMP gave little change in the Km. The apoenzyme which was quite unstable was stabilized by the addition of pyridoxal phosphate to a similar degree to the holoenzyme. The AMP which effectively protected the holoenzyme against various treatments of inactivation exhibited little protection for the apoenzyme. When proteolytic digestibility was examined, AMP exhibited marked protection for the holoenzyme; whereas the addition of pyridoxal phosphate to the holoenzyme gave no protection. Pyridoxal phosphate in the threonine deaminase probably not only participates in the catalytic reaction as a cofactor but also contributes to the maintenance and stabilization of the proper conformation of the enzyme protein presumably in a way different from that of AMP.