Recombinant human tyrosine hydroxylase isozymes

Abstract

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 Escherichiu 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 (1 3 -40-fold) by incubation with Fe(I1) salts (concentration of iron at half-maximal activation = 6-14 pM), and were inhibited by other divalent metal ions, e.g. Zn(II), Co(I1) and Ni(I1). They all bind stoichiometric amounts of Fe(I1) and Zn(I1) with high affinity (Kd = 0.2-3 pM at pH 5.4-6.5). Similar time courses were observed for binding of Fe(I1) and enzyme activation. In the absence of any free Fe(I1) 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.Tyrosine 3-monooxygenase (tyrosine hydroxylase) is a tetrahydropteridine-dependent enzyme which catalyses the rate-limiting step in the biosynthesis of catecholamines [ 11. Early studies showed that the enzyme activity is stimulated by ferrous iron and inhibited by certain iron chelators [2 -51.Subsequently, it was shown that the enzyme isolated from various tissues contained significant amounts of iron [6 -81, which seems to be involved in oxygen activation, an essential step in the catalytic reaction [6-81. However, partly due to the difficulties involved in obtaining pure tyrosine hydroxylase, no systematic study has been performed in order to determine the metal-binding properties of this enzyme.The recent expression of cloned tyrosine hydroxylase in high yield in eukaryotic cells using the baculovirus system [9, 101 and in prokaryotic cells [ 10 a] has, however, changed this situation. Human tyrosine hydroxylase exists as four different isozymes (TH1-TH4), generated through alternative splicing of pre-mRNA [ l l -131. Since these isozymes have different amino acid sequences in their regulatory N-terminal region and different distribution in the nervous system [14], it has been postulated that the occurrence of multiple molecular forms of the enzyme has important functional consequences. Previous studies, in which the isozymes have been expressed in low quantities in eukaryotic systems, have also demonstrated different kinetic properties of the various isozymes [15, 161. Correspondence to J. Haavik, Department of Biochemistry, UniAbbreviations. TH 1 -TH4, tyrosine hydroxylase isozymes 1 -4. Enzyme. Tyrosine 3-monooxygenase or tyrosine hydroxylase versity of Bergen, N-5009 Berge...

Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation

“…3B). The stoichiometry of ['4C]dopamine binding to the enzyme was the same in the presence of a twofold excess of Fe(I1) (data not shown), thus confirming the proposed 1 : 1 : 1 ratio of the dopamine/ iron/enzyme complex [11,13,161.…”

“…mg-' (hTH1 and hTH4)and0.2 pmol . min-' mg-'(hTH2)at3O0C [11]. The rate of phosphorylation of hTHl and hTH2 was measured in incubation mixtures containing 10 mM MgC12, 0.1 mM or 0.5 mM [32P]ATP and 40 mM Hepes, pH 7.0.…”

Three-way Interaction between 14-3-3 Proteins, the N-terminal Region of Tyrosine Hydroxylase, and Negatively Charged Membranes

“…Expression and Purification of Proteins-Recombinant human TH, isoform 1 (isoform b; NCBI accession code NP_ 000351.2; hTH1) was expressed in Escherichia coli and purified to homogeneity by heparin-Sepharose (Amersham Biosciences) chromatography as described (39). Phosphorylation of hTH1 at Ser 19 by p38 regulated/activated protein kinase (PRAK) was performed as previously described (17), obtaining a stoichiometry of 0.8 phosphate incorporated/subunit of hTH1.…”

Regulation of tyrosine hydroxylase by stress‐activated protein kinases

“…Catalase, dithiothreitol (DTT) and sodium dodecyl sulphate (SDS) were from Sigma (St Louis, MO, USA), tyrosine from Serva (Heidelberg, Germany), Zwittergent from Calbiochem (La Jolla, CA, USA) and trypsin from Boehringer Mannheim (Mannheim, Germany). hTH isoform 1 (hTH1) was expressed in Escherichia coli and purified as in Haavik et al (1991). The yeast (Saccharomyces cerevisiae) 14-3-3 protein BMH1 (6 · histidine tagged) and sheep brain 14-3-3 were expressed and purified as described in (Kleppe et al 2001).…”