Copper proteins phenylalanine hydroxylase

Tyrosinase is a monooxygenase which catalyzes the incorporation of one oxygen atom from dioxygen into phenols and further oxidizes the catechols formed to o-quinones (oxidase action). A comparison of spectral (EPR, electronic absorption, CD, and resonance Raman) properties of oxy-tyrosinase and its derivatives with those of oxy-Hc establishes a close similarity of the active site structures in these proteins (26-29). Thus, it seems likely that there is a close relationship between the binding of dioxygen and the ability to "activate" it for reaction and incoiporation into organic substrates. Other important copper monooxygenases which are however of lesser relevance to the model studies discussed below include dopamine p-hydroxylase (16,30) and a recently described copper-dependent phenylalanine hydroxylase (31). 

These systems are also described as normal copper proteins due to their conventional ESR features. In the oxidized state, their color is light blue (almost undetectable) due to weak d-d transitions of the single Cu ion. The coordination sphere around Cu, which has either square planar or distorted tetrahedral geometry, contains four ligands with N and/or 0 donor atoms [ 12, 22]. Representative examples of proteins with this active site structure (see Fig. 1) and their respective catalytic function include galactose oxidase (1) (oxidation of primary alcohols) [23,24], phenylalanine hydroxylase (hydroxy-lation of aromatic substrates) [25,26], dopamine- 6-hydroxylase (C-Hbond activation of benzylic substrates) [27] and CuZn superoxide dismutase (disproportionation of 02 superoxide anion) [28,29]. 

Two phenylalanine hydroxylases have been described, one containing iron and one containing copper. Both are phylogenetically related to the group of enzymes which hydroxylate aromatic amino acids, including tryptophane and tyrosine hydroxylase [187]. No phylogenetic relationship could be constructed to other proteins [183-186,327]. 

The process occurring here is reminiscent of the N.I.H. shift, which is well known to occur in iron hydroxylases such as cytochrome P-450 and mammalian PAH [1,167], For example, action of PAH on [4-3H]phenylalanine produces >90% [3-3H]tyrosine. Here, a presumed electrophilic iron-oxy species produces a carbonium ion intermediate from which a 1,2-shift occurs, giving a resonance stabilized cation rearomatization through loss of H+ (or 3H+) gives the observed product as a result of a heavy atom isotope effect. Thus, it appears that the N.I.H. shift mechanism for copper has been discovered for a chemical model system prior to its observation in proteins. 

The copper metalloenzymes are involved in oxygen-using reactions. These enzymes include cytochrome c oxidase (respiratory chain), lysyl oxidase (collagen synthesis), and dopamine [3-hydroxylase (neurotransmitter synthesis). Lysyl oxidase is a small protein with a molecular weight of 32 kDa. This enzyme contains an unusual modification, namely cross-linking between two different parts of its polypeptide chain. The cross-linked region consists of a structure called lysine tyrosylquinone (Klinman, 1996). Two amino acids are involved in this cross-linked structure, and these are Lys 314 and Tyr 349. Lysine tyrosylquinone is used as a cofactor and is necessary for the catalytic activity of the enzyme. Other copper metalloenzymes contain a related cofactor, namely 2,4,5-tiihydrox5q5henylalanine (topaquinone, TPQ). Serum amino oxidase is a copper metalloenzyme that contains TPQ. TPQ consists of a modified residue of phenylalanine. The copper in the active site of the enzyme occurs immediately adjacent to the TPQ cofactor. 

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