On the role of pyridoxal phosphate for AMP-dependent threonine deaminase of Escherichia coli

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    Department of Medical Chemistry, Kyoto University Faculty of Medicine

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  • ON THE ROLE OF PYRIDOXAL PHOSPHATE FOR AMP-DEPENDENT THREONINE DEAMINASE OF <i>ESCHERICHIA COLI</i>

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1. Threonine deaminase of anaerobically grown Escherichia coli was purified about 125-fold by sonic disintegration, streptomycin treatment, ammonium sulfate fractionation, Sephadex G-200 gel fractionation, and DEAE-Sephadex column chromatography. Inclusion of AMP and β-mercaptoethanol at 5mM 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 404mμ. The enzyme also exhibited a fluorescence spectrum with a maximum emission at 510mμ upon activation at 420mμ.<br>2. Resolution of pyridoxal phosphate from the threonine deaminase was performed by hydroxylamine treatment followed by gel filtration. For the apoenzyme, pyridoxal phosphate was the most effective reactivator, and pyridoxamine phosphate was slightly effective. Pyridoxal, pyridoxamine, and pyridoxine were without effect. Km for pyridoxal phosphate was 7.7μM, and the presence of AMP gave little change in the Km.<br>3. The apoenzyme was quite unstable and was stabilized by the addition of pyridoxal phosphate to a similar degree to the holoenzyme. AMP, which protected the holoenzyme effectively 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.<br>The above results suggest that pyridoxal phosphate in the threonine deaminase 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.

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