Rat Liver Phenylalanine Hydroxylase, an Iron Enzyme

Abstract

Abstract Phenylalanine hydroxylase that is essentially pure contains 1 to 2 moles of iron per mole of enzyme (assuming a molecular weight of 100,000). Electron spin resonance (ESR) studies have shown that the iron is present in the high spin ferric form. The addition of substrates (i.e. phenylalanine and dimethyltetrahydropterin) causes the signal for this form to disappear. The iron, 50 to 80%, can be removed by treatment of sucrose gradient purified enzyme with o-phenanthroline and cysteine; the loss of iron leads to the loss of enzymatic activity. The enzyme activity is restored by FeCl2 with half-maximum restoration achieved at 5 x 10-5 m FeCl2. Mercurous, nickelous, cobalt, manganese, cupric, chromium, cadmium, and zinc ions are ineffective in restoring activity to the apoenzyme (o-phenanthroline-treated enzyme). As judged by gel electrophoresis, the apoenzyme has the same charge and molecular weight as the holoenzyme. The apoenzyme, however, is much less stabile at 25° than the holoenzyme; FeCl2 restored the original stability to the apoenzyme. Furthermore, the apoenzyme has only three free cysteines per 50,000 molecular weight subunit, whereas the holoenzyme has five cysteines per subunit. The amino acid composition of the enzyme was also determined.