A CHIMERIC TYROSINE TRYPTOPHAN HYDROXYLASE - THE TYROSINE-HYDROXYLASEREGULATORY DOMAIN SERVES TO STABILIZE ENZYME-ACTIVITY/

The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-termina l regulatory domain and a carboxyl-terminal catalytic domain. A chimer ic hydroxylase was generated by coupling the regulatory domain of TH ( TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recomb inant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in mH because this residue is within a cons erved five amino-acid span (ValProTrpPhePro) that defines the beginnin g of the highly homologous catalytic domains of TH and TPH. Radioenzym atic activity assays demonstrated that the TH-R/TPH-C chimera hydroxyl ates tryptophan, but not tyrosine. Therefore, the regulatory domain do es not confer substrate specificity. Although the TH-R/TPH-C enzyme di d serve as a substrate for protein kinase (PKA), activation was not ob served following phosphorylation. Phosphorylation studies in combinati on with kinetic data provided evidence that TH-R does not exert a domi nant influence on TPH-C. Stability assays revealed that, whereas TH ex hibited a t(1/2) of 84 min at 37 degrees 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 dele tion of 165 amino acids from the N-terminus) of TH rendered the cataly tic domain highly unstable, as demonstrated by a t(1/2) of 14 min. The authors conclude that the regulatory domain of TH functions as a stab ilizer of enzyme activity. As a corollary, the well-characterized inst ability of TPH may be attributed to the inability of its regulatory do main to stabilize the catalytic domain.