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 (6 R )‐ l erythro ‐5,6,7,8‐tetrahydrobiopterin (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 μ m ). On phosphorylation of Ser40 by protein kinase A, the affinity for H 4 biopterin increased ([ S ] 0.5  = 11 ± 2 μ m ) and the negative cooperativity was amplified ( h  = 0.27 ± 0.03). The dimeric C‐terminal deletion mutant (Δ473–528) of hTH1 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 ± 5.7 μ m ) and 3‐methyl‐5,6,7,8‐tetrahydropterin (H 4 3‐methyl‐pterin)( h  = 1.0 ± 0.1; K m  = 687 ± 50 μ m ). In the presence of 1 m m H 4 3‐methyl‐pterin, used as a competitive cofactor analogue to BH 4 , hyperbolic saturation curves were also found for 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 (ΔRU) response with negative cooperativity ( h  = 0.52–0.56). Infrared spectroscopic studies revealed a reduced thermal stability both of the apo‐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.