Regulatory Properties of the Tetrahydropterin Cofactor in the Reaction Catalysed by Human Tyrosine Hydroxylase Isoforms 1–4

Abstract

The interaction of tyrosine hydroxylase (TH, EC 1.14.16.2), 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.