Hydroxylase reaction

Figure28-10. The phenylalanine hydroxylase reaction. Two distinct enzymatic activities are involved. Activity II catalyzes reduction of dihydrobiopterin by NADPH, and activity I the reduction of O2 to HjO and of phenylalanine to tyrosine. This reaction is associated with several defects of phenylalanine metabolism discussed in Chapter 30.

Figure 28-11. The prolyl hydroxylase reaction. The substrate is a proline-rich peptide. During the course of the reaction, molecular oxygen is incorporated into both succinate and proline. Lysyl hydroxylase catalyzes an analogous reaction.

Myllyla, R., Schubotz, L.M., Weser, U. and Kivirikko, K.I. (1979). Involvement of superoxide in the prolyl and lysyl hydroxylase reactions. Biochem. Biophy. Res. Commun. 89, 98-102. 

In order to function, CYP-450 enzymes require a source of reducing power (shown as 2H+ for the hydroxylase reaction above) in the form of NADPH. This coenzyme is a... 

The following is review on the molecular and physical properties of this class of monooxygenases, which are also known as hydroxylases. A typical monooxygenase reaction is the hydroxylation of an alkane to an alcohol which involves a reduced cosubstrate that reduces a second atom within the O2 molecule to form water. Flavin-containing monooxygenases include lysine oxygenase and 4-hydroxybenzoate hydroxylase. Reduced pteri-dines are involved in the phenylalanine hydroxylase and tryptophan hydroxylase reactions. See also Cytochrome P-450... 

In the normal prolyl 4-hydroxylase reaction (Fig. 4a), one molecule of a-ketoglutarate and one of 02 bind to the enzyme. The a-ketoglutarate is oxidatively decarboxylated to form C02 and succinate. The remaining oxygen atom is then used to hydroxylate an appropriate Pro residue in procollagen. No ascorbate is needed in this reaction. However, prolyl 4-hydroxylase also catalyzes an oxidative decarboxylation of a-ketoglutarate that is not coupled to proline hydroxylation—and this is the reaction that requires ascorbate (Fig. 4b). During this reaction, the heme Fe2+ becomes oxidized, and the oxidized form of the enzyme is inactive—unable to hydroxylate proline. The ascorbate consumed in the reaction presumably functions to reduce the heme iron and restore enzyme activity. 

FIGURE 18-24 Role of tetrahydrobiopterin in the phenylalanine hydroxylase reaction. The H atom shaded pink is transferred directly from C-4 to C-3 in the reaction. This feature, discovered at the NIH, is called the NIH Shift. 

Figure 19-2. Aromatic amino acid hydroxylase reaction. Aromatic amino acids are hy-droxylated by a common mechanism catalyzed by a family of hydroxylases.The enzyme family consists of phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase. In addition to substrate, all three enzymes require molecular oxygen and the cofactor tetrahydrobiopterin.Tetrahydrobiopterin is consumed in this reaction and converted into pterin 4cx-carbinolamine. DOPA, dihydroxyphenylalanine.

First two complexes with a (p.-oxo)(p-hydroxo)diiron (III) core [Fe2(0)(0H)(6TLA)2(C104)3] (I) and [Fe2(0)2(6TLA)2(C104)2] (II), were isolated and characterized (Zang et al 1995). Structure of a (-l,2-peroxo)bis(-carboxylato)diiron(m)model for the peroxo intermediate in the methane monooxygenase hydroxylase reaction cycle is presented in Fig, 6.3. 

Hydroxy-ABA (53) has been found in the leaves of Vicia faba6SS and other plants. 9 -Hydroxy-ABA (54) has been found in Brassica napus siliques along with its isomerized catabolite neophaseic acid (55),686 which had been formerly known as spi-phaseic acid.687 The occurrence of 7 - and 9 - hydroxy-ABAs is restricted to several plants at present, suggesting that these hydroxy-ABAs are minor catabolites of ABA. ABA 7 - and 9 -hydroxylases would be different from ABA 8 -hydroxylase since 7 - and 9 -hydroxy-ABAs are not detected in the products of ABA 8 -hydroxylase reaction. 

Kim, K., and Lippard, S. J., 1996, Structure and M ssbauer spectrum of a (p-l,2-peroxo)-bis( r-carboxylato)diiron(III) model for the peroxo intermediate in the methane monooxygenase hydroxylase reaction cycle, J. Am. Chem. Soc. 118 4914n4915. 

Catechol as an Activator of Tyrosinase. The phenolase activity of tyrosinase has been studied less completely than the catecholase activity, partly because of the lack of a satisfactory assay procedure. The phenolase reaction, however, is characterized by a lag time which can be abolished by adding dihydroxyphenylalanine (DOPA), the immediate product of the hydroxylation reaction 29S4, 102, 117), This phenomenon has been described by several investigators (29-34) and is illustrated in Figure 12, from Pomerantz and Warner (117), using the enzyme from Hamster melanoma. The same phenomenon has been analyzed by Duckworth and Coleman (102) for the mushroom enzyme. In the absence of DOPA, maximum velocity of the hydroxylase reaction is not reached for several minutes. Pomerantz and Warner (117) devised a convenient assay for the phenolase reaction by determining the radio-... 

Low serum calcium ion stimulates parathyroid glands to secrete PTH, which in turn stimulates 1-hydroxylase and inhibits 24-hydroxylase reactions in kidney. 

The alternate mechanism proposed by Hamilton59) seems more attractive since it is equivalent to the mechanism proposed for the prolyl hydroxylase reaction discussed earlier. 

Occasionally, the cytochrome P-450 system converts some chemieals to reactive species with carcinogenic potential (e.g., polycyclic hydrocarbons). The hepatic microsomal cytochrome P-450 system is inducible by many of its substrates. The cytoehrome P-450 of adrenal cortical mitochondria is involved in steroid hydroxylase reactions, and this system contains iron-sulfur (Fc2S2) proteins. 

The phenylalanine hydroxylase reaction is complex, occuring principally in liver but also in kidney. The hy-droxylating system is present in hepatocyte cytosol and contains phenylalanine hydroxylase, dihydropteridine reductase, and tetrahydrobiopterin as coenzyme. The hy-droxylation is physiologically irreversible and consists of a coupled oxidation of phenylalanine to tyrosine and of tetrahydrobiopterin to a quinonoid dihydroderivative with molecular oxygen as the electron acceptor ... 

NADH exhibits a lower and higher V ,ax for the reductase than NADPH. Thus, the pterin coenzyme functions stoichiometrically (in the hydroxylase reaction) and catalytically (in the reductase reaction). Deficiency of dihydropteridine reductase causes a substantial decrease in the rate of phenylalanine hy-droxylation. Dihydropteridine reductase and tetrahydrobiopterin are involved in hydroxylation of tyrosine and of tryptophan to yield neurotransmitters and hormones (dopamine, norepinephrine, epinephrine, and serotonin). Unlike phenylalanine hydroxylase, dihydropteridine... 

The rate-controlling step of catecholamine synthesis is the tyrosine hydroxylase reaction, for which the catecholamines are allosteric inhibitors. The enzyme is activated by the cAMP-dependent protein kinase phos-phorylating system. a-N-Methyl-p-tyrosine is an inhibitor of this enzyme and is used to block adrenergic activity in pheochromocytoma (see below). 

Fates of tyrosine. Tyrosine can be degraded by oxidative processes to ace-toacetate and fumarate which enter the energy generating pathways of the citric acid cycle to produce ATP as indicated in Figure 38-2. Tyrosine can be further metabolized to produce various neurotransmitters such as dopamine, epinephrine, and norepinephrine. Hydroxylation of tyrosine by tyrosine hydroxylase produces dihydroxyphenylalanine (DORA). This enzyme, like phenylalanine hydroxylase, requires molecular oxygen and telrahydrobiopterin. As is the case for phenylalanine hydroxylase, the tyrosine hydroxylase reaction is sensitive to perturbations in dihydropteridine reductase or the biopterin synthesis pathway, anyone of which could lead to interruption of tyrosine hydroxylation, an increase in tyrosine levels, and an increase in transamination of tyrosine to form its cognate a-keto acid, para-hydroxyphenylpyruvate, which also would appear in urine as a contributor to phenylketonuria. 

Obviously, more work is required to further substantiate the presence of the proposed radical intermediates in the p-hydroxybenzoate hydroxylase reaction, possibly via EPR and spin-trapping studies. Studies by Detmer and Massey 247) on phenol hydroxylase have indicated that the reaction rate constants for the conversion of meta-substituted substrates plotted versus the Hammett parameters yield a straight line of slope equal to 0.5. This is consistent with the mechanism proposed by Anderson, as the negative slope is expected for an electrophilic aromatic substitution reaction, while the small magnitude of the slope may be indicative of a radical mechanism. Furthermore, recent work by Massey and co-workers on p-hydroxybenzoate hydroxylase utilizing 6-hydroxy-FAD as cofactor and p-aminobenzoate as substrate indicated that the absorption spectrum of intermediate 67 exhibited a satellite band at 440 nm 248). Anderson et al. suggest that the satellite band may result from the formation of an aromatic phenoxyl radical at the C-6 position of the isoalloxazine ring of the flavin 244). This species would result from a shift of the initial peroxyl radical center from C(4a) to C-6 via N(5) 245). 

The physiological function of cytochrome is still uncertain, although several possibilities have been suggested. It has been implicated in fatty acid desaturation reactions in the endoplasmic reticulum of liver (II5) and in some hydroxylase reactions both in the reticulum 120) and mitochondria 121,122). It may participate in the reduction of methemo-globin in erythrocytes 109) since ferrocytochrome is readily oxidized by methemoglobin as well as ferricytochrome c. The half-life of cytochrome hi in rat liver microsomes is reported to be 45 hr 123). 

GABA synthesis, which depend on a functioning TCA cycle, are decreased as a result of elevated NADH levels, which inhibit TCA cycle enzymes. NADH levels are increased when oxygen is unavailable to accept electrons from the electron transport chain and NADH cannot be converted back into NAD. Even the synthesis of catecholamine neurotransmitters may be decreased because the hydroxylase reactions require O2. 

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