title: Catalytic core of rat tyrosine hydroxylase: terminal deletion analysis of bacterially expressed enzyme.
abstract: Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in catecholamine biosynthesis. This enzyme is hypothesized to consist of an amino-terminal regulatory domain and a carboxy-terminal catalytic domain. In the present studies, we have utilized recombinant DNA techniques to map the boundaries of the regulatory and catalytic domains of TH. We have isolated a full-length cDNA clone for rat pheochromocytoma TH and have expressed the enzyme in bacteria. Utilizing this bacterial expression system and polymerase chain reaction technology, we have constructed and subcloned genes for five amino-terminal deletion mutants (N delta 40, N delta 155, N delta 165, N delta 175 and N delta 200; N delta denotes amino-terminal deletion and the numerical value denotes the number of amino acids deleted) and two carboxy-terminal deletion mutants (C delta 19 and C delta 50). The catalytic core of rat tyrosine hydroxylase has been identified to include the region from amino acid #165 to amino acid #479. The amino-terminal deletion mutants, N delta 40, N delta 155 and N delta 165 are from 1.85 to 2.5-fold more active than unmodified recombinant TH, while the removal of 19 amino acids from the C-terminus (C delta 19) results in a 70% reduction in enzyme activity. Removal of additional sequences (ten more residues from the N-terminus [N delta 175]; or an additional 31 amino acids from the C-terminus [C delta 50]) results in protein that is totally without enzyme activity. As expected, removal of 40 (or more) N-terminal amino acids abolishes the ability of the catalytic subunit of the cAMP-dependent protein kinase to phosphorylate the recombinant enzyme; serine-40 is the phosphorylation site on TH for PKA. We conclude that the N-terminal boundary for the TH catalytic domain resides between residues 165 and 175 and that removal of this N-terminal domain (totally or partially) increases the activity of the enzyme. These findings confirm previous reports that proteolytic cleavage at amino acid #158 produces an active (and activated) catalytic fragment.
authors.affiliation: Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157-1016.
authors.forename: S J
authors.initials: SJ
authors.lastname: Walker
authors.forename: X
authors.initials: X
authors.lastname: Liu
authors.forename: R
authors.initials: R
authors.lastname: Roskoski
authors.forename: K E
authors.initials: KE
authors.lastname: Vrana
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000302:isolation & purification; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D014446:Tyrosine 3-Monooxygenase