Site-directed mutagenesis of serine 40 of rat tyrosine hydroxylase. Effects of dopamine and cAMP-dependent phosphorylation on enzyme activity.
Rat tyrosine hydroxylase expressed with a baculovirus expression system contains covalent phosphate and has kinetic parameters consistent with those expected of phosphorylated enzyme (Fitzpatrick, P. F., Chlumsky, L. J., Daubner, S. C., and O'Malley, K. L. (1990) J. Biol. Chem. 265, 2042-2047). The phosphorylation site was identified as serine 40, by purifying the enzyme from cells grown in the presence of [32P]phosphate. Replacement of serine 40 with alanine by site-directed mutagenesis prevented phosphorylation but had little effect on the steady-state kinetic parameters at pH 7. Both wild type and S40A tyrosine hydroxylase were expressed in Escherichia coli; the kinetic parameters of the enzymes purified from bacteria were nearly identical to those of the enzymes expressed with the baculovirus system, although the bacterially expressed enzyme contained no covalent phosphate. Treatment of this wild type enzyme with cAMP-dependent protein kinase decreased the KBH4 value about 2-fold but had no effect on the Vmax value at pH 7. Treatment with a stoichiometric amount of dopamine decreased the Vmax value 15-fold and increased the KBH4 value 2-3-fold. Phosphorylation of the dopamine-bound enzyme increased the Vmax value 10-fold and decreased the KBH4 value 2-fold. The kinetic parameters of the dopamine-bound recombinant enzyme were identical to those of enzyme purified from PC12 cells. In contrast, the S40A enzyme was converted to a less active form by treatment with dopamine but was not affected by phosphorylating conditions. These results are consistent with a model in which the major effect of phosphorylation of serine 40 is to relieve tyrosine hydroxylase from the inhibitory effects of catecholamines.
Department of Biochemistry and Biophysics, Texas A & M University, College Station 77843.
S C
SC
Daubner
C
C
Lauriano
J W
JW
Haycock
P F
PF
Fitzpatrick
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA; D004298:Dopamine / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D012694:Serine / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism*
D012694:Serine; D004247:DNA; D000242:Cyclic AMP; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine