Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation. Structure/activity studies and mechanistic implications.
Three isozymes of human tyrosine hydroxylase (hTH1, hTH2 and hTH4) were expressed in Escherichia coli and purified to homogeneity. Natural catecholamines and related synthetic compounds were found to be potent inhibitors, competitive to the tetrahydrobiopterin cofactor, of all the isozymes. Combining visible spectroscopy and equilibrium-binding studies, it was found that catecholamines bind to hTH1 and hTH2 with a stoichiometry of about 1.0 mol/mol enzyme subunit, interacting with the catalytic iron at the active site. All the isozymes tested were excellent substrates for cAMP-dependent protein kinase (Km = 5 microM, Vmax = 9.5 mumol.min-1.mg kinase-1). The incorporation of about 1.0 mol phosphate/subunit at Ser40 decreased the affinity of dopamine binding by a factor of 10. Conversely, the addition of stoichiometric amounts of Fe(II) and dopamine to the apoenzymes reduced both the affinity and stoichiometry of phosphorylation by cAMP-dependent protein kinase by 2-3-fold. These data provide evidence for a mutual interaction between the presumed regulatory and catalytic domains of hTH, and show that activation of the enzyme by phosphorylation and inactivation by binding of catecholamines are related events, which probably represent important mechanisms for the regulation of the enzyme activity in vivo.
Department of Biochemistry, University of Bergen, Norway.
B
B
Almås
B
B
Le Bourdelles
T
T
Flatmark
J
J
Mallet
J
J
Haavik
D001665:Binding Sites; D001667:Binding, Competitive; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D002395:Catecholamines / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007501:Iron / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002395:Catecholamines; D007527:Isoenzymes; D001708:Biopterins; D000242:Cyclic AMP; D007501:Iron; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; C003402:sapropterin; D004298:Dopamine