A chimeric tyrosine/tryptophan hydroxylase. The tyrosine hydroxylase regulatory domain serves to stabilize enzyme activity.
The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t1/2 of 84 min at 37 degrees C, TPH was much less stable (t1/2 = 28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by a t1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain.
Mockus
S M
SM
Program in Neuroscience, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC, USA.
Kumer
S C
SC
Vrana
K E
KE
D000818:Animals; D015394:Molecular Structure; D008970:Molecular Weight; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D014365:Tryptophan Hydroxylase / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014446:Tyrosine 3-Monooxygenase; D014365:Tryptophan Hydroxylase