Recombinant human tyrosine hydroxylase isozymes. Reconstitution with iron and inhibitory effect of other metal ions.
Human tyrosine 3-monooxygenase (tyrosine hydroxylase) exists as four different isozymes (TH1-TH4), generated by alternative splicing of pre-mRNA. Recombinant TH1, TH2 and TH4 were expressed in high yield in Escherichia coli. The purified isozymes revealed high catalytic activity [when reconstituted with Fe(II)] and stability at neutral pH. The isozymes as isolated contained 0.04-0.1 atom iron and 0.02-0.06 atom zinc/enzyme subunit. All three isozymes were rapidly activated (13-40-fold) by incubation with Fe(II) salts (concentration of iron at half-maximal activation = 6-14 microM), and were inhibited by other divalent metal ions, e.g. Zn(II), Co(II) and Ni(II). They all bind stoichiometric amounts of Fe(II) and Zn(II) with high affinity (Kd = 0.2-3 microM at pH 5.4-6.5). Similar time courses were observed for binding of Fe(II) and enzyme activation. In the absence of any free Fe(II) or Zn(II), the metal ions were released from the reconstituted isozymes. The dissociation was favoured by acidic pH, as well as by the presence of metal chelators and dithiothreitol. The potency of metal chelators to remove iron from the hydroxylase correlated with their ability to inhibit the enzyme activity. These studies show that tyrosine hydroxylase binds iron reversibly and that its catalytic activity is strictly dependent on the presence of this metal.
Department of Biochemistry, University of Bergen, Norway.
J
J
Haavik
B
B
Le Bourdelles
A
A
Martinez
T
T
Flatmark
J
J
Mallet
D002413:Cations, Divalent; D003001:Cloning, Molecular; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007502:Iron Chelating Agents / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008670:Metals / Q000494:pharmacology*; D011485:Protein Binding; D012322:RNA Precursors / Q000235:genetics; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013053:Spectrophotometry; D014446:Tyrosine 3-Monooxygenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D002413:Cations, Divalent; D007502:Iron Chelating Agents; D007527:Isoenzymes; D008670:Metals; D012322:RNA Precursors; D011994:Recombinant Proteins; D007501:Iron; D014446:Tyrosine 3-Monooxygenase