Regulatory Properties of the Tetrahydropterin Cofactor in the Reaction Catalysed by Human Tyrosine Hydroxylase Isoforms 1–4
The interaction of tyrosine hydroxylase (TH, EC 22.214.171.124), the rate-limiting enzyme of the catecholamine (CA) biosynthetic pathway, with its cofactor 6-(R)-5,6,7,8-tetrahydrobiopterin (BH4) seems to be highly specific. However, the dihydroxypropyl group in the Co-position of BH4 can be substituted with only moderate changes in kinetic parameters (1). TH is highly regulated, and many of the regulatory mechanisms (phosphorylation, feed-back inhibition) involve a change in the affinity for the cofactor. The four isoforms of human TH (hTH1–4) differ only in the length of their N-terminal regulatory domain (2). They all exist as homotetramers, containing a tetramerization domain in the C-terminal end. All isoforms have been expressed in E. coli and are isolated as nonphosphorylated apoenzymes with no CAs bound. The human recombinant enzyme thus represents a well defined system for structural and functional studies of the binding of BH4 to TH. We have previously reported a negative cooperativity in the binding of BH4 to hTH1 (Hill coefficient 0.39 < h < 0.58). This was modulated by phosphorylation by protein kinase A (h = 0.27 ± 0.03) (3). Time-course studies of the enzymatic reaction also demonstrated a time dependent change (burst) in activity, with a half-life of about 20 s for the kinetic transient (3). In this study we have further investigated the regulatory properties of BH4 with all human TH isoforms. Using synthetic cofactors as well as limited proteolysis, the relationship between the negative cooperativity and the kinetic transient in hTH1 was further characterised.
KeywordsHydroxyl Dopamine Tyrosine Trypsin Folate
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