Catalytic core of rat tyrosine hydroxylase: terminal deletion analysis of bacterially expressed enzyme

Stephen J. Walker, Xuan Liu, Robert Roskoski, Kent E. Vrana

Research output: Contribution to journalArticlepeer-review

29 Scopus citations

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 (NΔ40, NΔ155, NΔ165, NΔ175 and NΔ200; NΔ denotes amino-terminal deletion and the numerical value denotes the number of amino acids deleted) and two carboxy-terminal deletion mutants (CΔ19 and CΔ50). 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, NΔ40, NΔ155 and NΔ165 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 (CΔ19) results in a 70% reduction in enzyme activity. Removal of additional sequences (ten more residues from the N-terminus [NΔ175]; or an additional 31 amino acids from the C-terminus [CΔ50]) 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.

Original languageEnglish (US)
Pages (from-to)113-119
Number of pages7
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1206
Issue number1
DOIs
StatePublished - May 18 1994

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

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