Phenylalanine hydroxylase from Chromobacterium violaceum is a copper-containing monooxygenase. Kinetics of the reductive activation of the enzyme

Abstract

Pterin-dependent phenylalanine hydroxylase from Chromobacterium violaceum contains a stoichiometric amount of copper (Cu2+, 1 mol/mol of enzyme). Electron paramagnetic resonance spectroscopy of the enzyme indicates that it is a type II copper-containing protein. The oxidized enzyme must be reduced by a single electron to be catalytically active. Dithiothreitol was found to be an effective reducing agent for the enzyme. Electron paramagnetic resonance data and kinetic results indicate the formation of an enzyme-thiol complex during the aerobic reduction of the enzyme by dithiothreitol. 6,7-Dimethyltetrahydropterin also reductively activates the enzyme, but only in the presence of the substrate, and is kinetically less effective than dithiothreitol. The metal center is not reoxidized as a result of normal turnover. However, the data indicate an alternative pathway exists that results in slow reoxidation of the enzyme. The 4a-hydrate of 6-methyltetrahydropterin (4a-carbinolamine) is observed during turnover of the enzyme. This intermediate is also observed during the reaction catalyzed by the iron-containing mammalian enzyme, suggesting that the mechanism of oxygen activation is similar for both enzymes.

Phenylalanine Hydroxylase from Chromobacterium violaceum

“…The properties of the purified enzyme also differ from those described earlier (7,8,33). Prof. Benkovic has compared our results with those from his laboratory and concluded that two phenylalanine hydroxylases are present in C. violaceum.…”

EPR and 1H‐NMR spectroscopic studies on the paramagnetic iron at the active site of phenylalanine hydroxylase and its interaction with substrates and inhibitors

“…It has been shown by EPR spectroscopy that dithiothreitol reduces the catalytic Cu(I1) to Cu(1) in phenylalanine hydroxylase isolated from Chromobucterium violaceum [33] and it has been suggested that dithiothreitol also reduces the enzyme-bound ferric iron to the ferrous form in the rat enzyme [2]. However, our present EPR studies under aerobic conditions with the bovine enzyme (Fig.…”

Mechanism of Aromatic Amino Acid Hydroxylation

“…There is no evidence that the ferric enzyme is a catalytic intermediate; rather, these enzymes begin and end each catalytic cycle in the ferrous form. The C. violaceum enzyme has been isolated with copper instead of iron (20), and this observation led to the proposal that the bacterial enzyme does not require any metal for activity (21,22). More recently, C. violaceum PheH was shown to have an absolute requirement for iron for tyrosine formation (23), in agreement with the metal requirements of the eukaryotic enzymes.…”