Mutation of regulatory serines of rat tyrosine hydroxylase to glutamate: effects on enzyme stability and activity.
Tyrosine hydroxylase is phosphorylated at four serine residues in its amino-terminus by multiple kinases. Phosphorylation of serine 40 by cAMP-dependent protein kinase results in alleviation of dopamine inhibition [J. Biol. Chem. 267 (1992) 12639]. The other serines are at positions 8, 19, and 31. The effect of phosphorylation at these serines has been investigated using mutated forms of tyrosine hydroxylase containing glutamates at the positions of the serines. The S8E, S19E, and S31E tyrosine hydroxylase variants have similar steady-state kinetic parameters and similar binding affinity for catecholamines to wild-type enzyme. The S8E, S19E, S31E, and S40E variants differ in stability at elevated temperatures. The S40E variant is the least stable, while the others are all more stable than wild-type enzyme. The increased stability of S8E, S19E, and S31E tyrosine hydroxylases may be one of the physiological effects of phosphorylation. It may also have implications for the interpretation of activities of heterogeneous mixtures of tyrosine hydroxylase which have been phosphorylated.
Royo
Montserrat
M
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.
Fitzpatrick
Paul F
PF
Daubner
S Colette
SC
D000595:Amino Acid Sequence; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism / Q000494:pharmacology; D014644:Genetic Variation; D018698:Glutamic Acid / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009841:Oligonucleotides / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D012694:Serine / Q000737:chemistry*; D013053:Spectrophotometry; D013696:Temperature; D013997:Time Factors; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics* / Q000378:metabolism
D009841:Oligonucleotides; D018698:Glutamic Acid; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D017868:Cyclic AMP-Dependent Protein Kinases; D004298:Dopamine