Site-directed mutagenesis of tyrosine hydroxylase. Role of serine 40 in catalysis.
We have investigated the role of serine 40 (Ser-40) in tyrosine hydroxylase (TH) catalysis of basal and activated enzymes by protein kinase A (PKA)-mediated phosphorylation. Wild type and mutant TH were transiently and stably expressed in AtT-20 cells, and the enzymatic activities of the recombinant enzymes were analyzed. The specific enzymatic activity of transiently expressed TH mutants Ser-40-->leucine or-->tyrosine (Leu-40m or Tyr-40m) was higher than that of the wild type enzyme or of other mutants in which Ser-8, -19, and -31 were replaced by leucine. The kinetic studies carried out with the stably expressed TH show that the Km for the cofactor 6-methyltetrahydropterine is lower and the Ki for dopamine is higher when the enzymatic hydroxylation is catalyzed by the Leu-40m or Tyr-40m than by the wild type enzyme. The kinetic parameters and the pH profile of the enzymatic hydroxylation catalyzed by the Leu-40m or Tyr-40m are similar to the enzyme activated by PKA-mediated phosphorylation. We suggest that Ser-40 in TH exerts an inhibitory influence on the enzymatic activity, and its replacement with another amino acid by site-directed mutagenesis or its modification by phosphorylation leads to a change in conformation with an increased enzymatic activity. The importance of Ser-40 in the activation of TH by PKA-mediated phosphorylation was investigated by comparing the activation of the wild type enzyme with that of Leu-40m or Tyr-40m. The findings that the enzymatic activity is increased by PKA-mediated phosphorylation of the wild type enzyme, but not of the Leu-40m or Tyr-40m, demonstrate that phosphorylation at Ser-40 is essential for activation of TH by PKA. The findings that addition of ATP plus cAMP to homogenates from transfected AtT-20 cells stimulates the recombinant wild type TH activity indicate that these cells contain endogenous cAMP-dependent protein kinase.
New York University Medical Center, Department of Psychiatry, Millhauser Laboratories, New York.
J
J
Wu
D
D
Filer
A J
AJ
Friedhoff
M
M
Goldstein
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004789:Enzyme Activation; D015723:Gene Library; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016716:PC12 Cells; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D014162:Transfection; D014407:Tumor Cells, Cultured; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012694:Serine; D004247:DNA; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases