Tyrosine hydroxylase binds tetrahydrobiopterin cofactor with negative cooperativity, as shown by kinetic analyses and surface plasmon resonance detection

Kinetic studies of tetrameric recombinant human tyrosine hydroxylase isoform 1 (hTH1) have revealed properties so far not reported for this enzyme. Firstly, with the natural cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobioptrin (H(4)biopterin) a time-dependent change (burst) in enzyme activity was observed, with a half-time of about 20 s for the kinetic transient. Secondly, nonhyperbolic saturation behaviour was found for H(4)biopterin with a pronounced negative cooperativity (0.39 < h < 0.58: [S](0.5) = 24 +/- 4 mu M). On phosphorylation of Ser40 by protein kinase A, the affinity for H(4)biopterin increased ([S](0.5) = 11 +/- 2 mu M) and the negative cooperativity was amplified (h = 0.27 +/- 0.03), The dimeric C-terminal deletion mutant (Delta 473-528) of hTHI also showed negative cooperativity of H(4)biopterin binding (h = 0.4. Cooperativity was not observed with the cofactor analogues 6-methyl-5,6,7,8-tetrahydropterin (h = 0.9 +/- 0.1; K-m = 62.7 s 5.7 mu M) and 3-methyl-5,6,7,8-tetrahydropterin (H(4)3-methyl-pterin)(h = 1.0 +/- 0.1; K-m = 687 +/- 50 mu M) In the presence of 1 mM H(4)3-methyl-pterin, used as a competitive cofactor analogue to BH4, hyperbolic saturation curves were also found fur H(4)biopterin (h = 1.0), thus confirming the genuine nature of the kinetic negative cooperativity. This cooperativity was confirmed by real-time biospecific interaction analysis by surface plasmon resonance detection. The equilibrium binding of H(4)biopterin to the immobilized iron-free apoenzyme results in a saturable positive resonance unit (Delta RU) response with negative cooperativity (h = 0.52-0.56). Infrared spectroscopic studies revealed a reduced thermal stability both of the ape-and the holo-hTH1 on binding of H(4)biopterin and L-erythro-dihydrobiopterin (H(2)biopterin). Moreover, the ligand-bound forms of the enzyme also showed a decreased resistance to limited tryptic proteolysis. These findings indicate that the binding of H(4)biopterin at the active site induces a destabilizing conformational change in the enzyme which could be related to the observed negative cooperativity. Thus, our studies provide new insight into the regulation of TH by the concentration of H(4)biopterin which may have significant implications for the physiological regulation of catecholamine biosynthesis in neuroendocrine cells.