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A chimeric tyrosine/tryptophan hydroxylase

The tyrosine hydroxylase regulatory domain serves to stabilize enzyme activity

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Abstract

The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxylterminal 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°C, TPH was much less stable (t 1/2=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 at 1/2 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.

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Authors and Affiliations

  1. Program in Neuroscience, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC

    Susan M. Mockus

  2. Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC

    Sean C. Kumer & Kent E. Vrana

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  1. Susan M. Mockus
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  2. Sean C. Kumer
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Mockus, S.M., Kumer, S.C. & Vrana, K.E. A chimeric tyrosine/tryptophan hydroxylase. J Mol Neurosci 9, 35–48 (1997). https://doi.org/10.1007/BF02789393

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