Human tyrosine hydroxylase isoforms. Inhibition by excess tetrahydropterin and unusual behavior of isoform 3 after camp-dependent protein kinase phosphorylation.
Human tyrosine hydroxylase exists as four isoforms (hTH1-4), generated by alternative splicing of pre-mRNA, with tissue-specific distribution. Unphosphorylated hTH3 and hTH1 were produced in large amounts in Escherichia coli and purified to homogeneity. The phosphorylation sites were determined after labeling with [32P]phosphate in the presence of cAMP-dependent protein kinase (PKA) and calmodulin-dependent protein kinase II (CaM-PKII). Ser40 was phosphorylated by PKA, and both Ser19 and Ser40 were phosphorylated by CaM-PKII. The enzyme kinetics of hTH3 were determined in the presence of various concentrations of the natural co-substrate (6R)-tetrahydrobiopterin and compared with those of recombinant hTH1 (similar to rat TH). We show that, under initial velocity conditions, excess (6R)-tetrahydrobiopterin inhibits hTH3 and hTH1. The TH catalytic constants (kcat) were determined for each of the two isoenzymes: hTH3 is about five times more active than hTH1. Phosphorylation by CaM-PKII did not affect the kinetic parameters of hTH3. The classical activation of TH by PKA phosphorylation, demonstrated for hTH1, was not observed with hTH3. Furthermore, hTH3 escapes activity regulation by phosphorylation and is always more active than phosphorylated hTH1. The properties of the hTH3 enzyme may be relevant to diseases affecting dopaminergic cells.
Alterio
J
J
Laboratoire de Génétique Moleculaire de la Neurotransmission et des Processus Neurodégénératifs, Hôpital de la Pitié Salpêtrière, 75013 Paris, France.
Ravassard
P
P
Haavik
J
J
Le Caer
J P
JP
Biguet
N F
NF
Waksman
G
G
Mallet
J
J
D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011622:Pterins / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014446:Tyrosine 3-Monooxygenase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism
D007527:Isoenzymes; D011622:Pterins; D011994:Recombinant Proteins; C011552:tetrahydropterin; D014446:Tyrosine 3-Monooxygenase; D017868:Cyclic AMP-Dependent Protein Kinases