Mechanistic studies using beef liver enzyme and substrate specifically labelled with deuterium in the methylene group indicate that side-chain migration occurs with retention of configuration at the methylene group [B904]. Studies using an enzyme with unspecified origin, but presumably mammalian, have shown that the phenolic hydroxyl group is not exchanged during the reaction. This indicates that, although still possible, an intermediate with a peroxide 1,4-bridge is most unlikely [A844]. 1-Carboxymethyl-
1-hydroxy-4-oxocyclohexa-2,5-diene is not an intermediate [A346].
Human enzyme is a dimer, molecular weight 87000. It acts on the keto isomer with optima at pH 4.5 and 7.8. It is activated by reducing agents such as ascorbate and is very sensitive to inacti-vation by peroxide. Iron- and copper-chelating reagents are inhibitory, and reactivation by dialysis indicates that the chelators do not remove the metal from the enzyme molecule. It exists in three forms with different pI between 6.5 and 7.5. These appear to be dimers of two monomeric forms [A3128, A3129].
A genetic defect in mice leads to a deficiency of this enzyme [G216].
Pig enzyme is inhibited by pentafluorophenyl-pyruvate and thiophenyl oxalate. It is inactivated by tetrafluoro-4-hydroxyphenylpyruvate and by
2- and 3-thienylpyruvate [H382]. The enzyme is stable only after purification. During storage it polymerizes, and this is reversed by thiols. The molecular weight is 89 000 by ultracentrifugation, and 52 000 and 44 000, respectively, by gel filtration and electrophoresis. It contains Cu and Fe (not stoichiometric). The temperature coefficient was found to be unusually high, about five (even higher than that reported by Goodwin, B.L. (1972) Tyrosine Catabolism, Clarendon Press, Oxford). The activity is stimulated by some hydrophilic solvents, including alcohols and ethers [C208].
Rat liver enzyme, molecular weight 63 000 and pI 5.85, is inactivated by dialysis and other processes that remove small molecules, and is reactivated by Fe2+ and dichlorophenolindo-phenol [A2702]. At birth, about 25 per cent of the enzyme is in an active form, and this increases to
90 per cent in the adult [A771]. The molecular weight is 45 085 by mass spectrometry (theoretical value 45 082). It also shows a-oxoisocaproate dioxygenase activity. At the C-terminal a 14 amino acid sequence is essential for activity; a mutant with a deletion of these amino acids produces an inactive protein [J235].
Bamboo enzyme is highly specific, molecular weight about 10 000. It is stoichiometric, and no intermediate has been detected [B238]. Zea mays enzyme, molecular weight 43 000 and optimum pH 7.3, requires a reducing system or ascorbate and catalase [J247]. Its presence has also been reported in Drosophyllum lusitanicum [A1149].
Enzyme from Pseudomonas strain PJ874 is blue and contains both iron and zinc. It catalyzes a stoichiometric reaction involving the keto isomer of the substrate, with an optimum and maximal stability at pH 7.9. It appears to act by a mono-iso-ordered bi-bi mechanism, in which Fe3+ is reduced [C365, C728]. Another publication claims that enzyme from strain PJ874 is a tetramer, monomeric molecular weight 36 000, optimum pH 7 and pI 4.8, and contains both Fe and Cu. The Fe content parallels activity during purification and the ratio of activity towards p -hydroxyphenylpyruvate and phenylpyruvate remains constant [A3106].
Tryptophan 5-hydroxylase; (tryptophan 5-monooxygenase, E.C. 184.108.40.206)
+ O2 0 5-hydroxy-l-tryptophan
This reaction is a key step in the formation of the neurotransmitter serotonin, as well as the hormone melatonin, which responds to light and dark periods and is associated with the biological clock in vertebrates.
Activity in brain (presumably human) median and dorsal raphe nuclei is 50-100 times greater than in caudate nucleus and hippocampus [A1690].
Mouse gut mucosal enzyme is probably of enterochromaffin origin [E503]. Brain enzyme is activated by phosphorylation and inactivated by
Tryptamine 5-hydroxylation phosphatase [A3813]. Mouse mastocytoma enzyme, pI 6.0, has a molecular weight of 270 000, and despite a monomeric molecular weight of 53 000 it is claimed to be a tetramer. l-Phenylalanine and (marginally) l-tyrosine are also substrates [C216].
Rat brain stem enzyme, which is stimulated by Fe2 + , is composed of two isozymes, one of which has a molecular weight of 300 000. Despite a monomeric molecular weight of 59 000 it is also claimed to be a tetramer. l-Phenylalanine is a substrate, but not l-tyrosine [C113]. Influences in vivo that reduce brain tryptophan concentration cause the enzyme activity to increase, apparently by an increase in Vmax [A1920], an effect that should sustain brain serotonin levels in face of alterations in tryptophan availability. Liver enzyme (that also acts on phenylalanine) is stimulated by 5-fluorotryptophan and 7-azatryptophan by up to 20-fold, and at higher cofactor concentrations by phenylalanine and thienylalanine [A791]. Pineal enzyme shows a diurnal rhythm with raised activities at night, and this is eliminated in constant light. Cycloheximide but not actinomycin D causes a rapid loss in activity, which suggests that enzyme activity is controlled at the translation step. Sulphydryl compounds protect the enzyme from inactivation at 0° but not at higher temperatures, and dithiothreitol reactivates the inactivated enzyme. Rapid in vivo inactivation is caused by p -chlorophenylalanine, and this is reversed within 24h [A3237]. Presumably this diurnal rhythm is associated with the diurnal rhythm of melatonin concentrations.
Pig brain stem enzyme, molecular weight 55000-60 000, is not stimulated by Fe2+ or by chelating agents and is unstable during storage [A1306].
The activity of chick brain enzyme, optimum pH 7.8-8.0 [A3236], increases about 10-fold just before and after hatching [A3130].
Enzyme from yellowfin tuna resembles mammalian enzyme. It is a trimer, molecular weight 280 000 [H910]. Skipjack liver enzyme is a homotrimer, molecular weight 288 000 and optimum pH 8.0 [H394].
Sedum morgaianum enzyme has an optimum at pH 7.5 [D41].
Chromobacterium violaceum enzyme, optimum pH 7, is inducible by tryptophan and (better) phenylalanine. d-Tryptophan is also a substrate, but phenylalanine is not. It requires oxygen, reduced pteridine cofactor and a thiol. Both phenylalanine and p -chlorophenylalanine are inhibitory [A1597].
Peganum harmala enzyme acts on tryptamine, a-methyl-, N-methyl- and 6-fluorotryptamine [A1330, F9, G906].
Kynurenine 3-hydroxylase; (kynurenine 3-monooxygenase; E.C. 220.127.116.11)
l-Kynurenine 0 3-hydroxy-l-kynurenine
Studies on rabbit, rat, gerbil and mouse demonstrate that activity is found in liver, lung and brain, in decreasing order [J830].
Rat liver enzyme is associated with the mitochondrial outer membrane. When solubilized it can be separated into two fractions by chromatography; the major one has a molecular weight of 200 000, optimum pH 8 and pI 5.4. It contains dissociable FAD (one mol/mol) that cannot be replaced by FMN or riboflavin. It is activated by FAD, dithiothreitol and phosphatidylcholine, and maximal activity occurs in 10 mM KCl. Inhibition is brought about by p -chloromercuribenzoate and bathocuproinsulphonate [A2270, A2293, A2482].
Honeybee eye enzyme, which requires NADPH, has an optimum at pH 7.25 [A1064]. Drosophila melanogaster eye enzyme appears to be mitochondrial [A1194].
Anthranilate 3-hydroxylase; (anthranilate 3-monooxygenase; E.C. 18.104.22.168)
Anthranilate 0 3-hydroxyanthranilate
Aspergillus niger enzyme, molecular weight 43 000-45 000, optimum pH 8.2 and pI 5.36,
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