In the present study the optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, was used for real-time measurements of the reversible binding of the pterin cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and l-phenylalanine (l-Phe) to human phenylalanine hydroxylase (hPAH). When BH(4) (241 Da) was injected over the sensor chip with immobilized tetrameric wt-hPAH a positive DeltaRU response was observed with a square-wave type of sensorgram and a saturable response (about 25 RU/(pmol subunit/mm(2)) with a [S](0.5) value of 5.6 +/- 0.8 microM for the pterin cofactor. The rapid on-and-off rates were, however, not possible to determine. By contrast, when l-Phe (165 Da) was injected a time-dependent increase in RU (up to about 3 min) and a much higher saturable DeltaRU response (about 75 RU/(pmol subunit/mm(2)) at 2 mM l-Phe) than expected (i.e., <5 RU/(pmol subunit/mm(2))) from the low molecular mass of l-Phe were observed in the sensorgram. The half-time for the on-and-off rates were 6 +/- 2 and 9 +/- 1 s, respectively, at 2 mM l-Phe. The steady-state (apparent equilibrium) response revealed a hyperbolic concentration dependence with a [S](0.5) value of 98 +/- 7 microM. The [S](0.5) values of both pterin cofactor and l-Phe were lower than those determined by steady-state enzyme kinetic analysis. Evidence is presented that the DeltaRU response to l-Phe is accounted for by the global conformational transition which occurs in the enzyme upon l-Phe binding, i.e., by the slow reversible transition from a low activity state ("T"-state) to a high activity state ("R"-state) characteristic of this hysteretic enzyme.

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)-Lerythro-5,6,7, 8-tetrahydrobiopterin (H4biopterin) 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 H4biopterin with a pronounced negative cooperativity (0.39 < h < 0.58; [S]0.5 = 24 +/- 4 microM). On phosphorylation of Ser40 by protein kinase A, the affinity for H4biopterin increased ([S]0.5 = 11 +/- 2 microM) and the negative cooperativity was amplified (h = 0.27 +/- 0.03). The dimeric C-terminal deletion mutant (Delta473-528) of hTH1 also showed negative cooperativity of H4biopterin binding (h = 0.4). Cooperativity was not observed with the cofactor analogues 6-methyl-5,6,7,8-tetrahydropterin (h = 0.9 +/- 0.1; Km = 62.7 +/- 5.7 microM) and 3-methyl-5,6,7, 8-tetrahydropterin (H43-methyl-pterin)(h = 1.0 +/- 0.1; Km = 687 +/- 50 microM). In the presence of 1 mM H43-methyl-pterin, used as a competitive cofactor analogue to BH4, hyperbolic saturation curves were also found for H4biopterin (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 H4biopterin to the immobilized iron-free apoenzyme results in a saturable positive resonance unit (DeltaRU) 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 H4biopterin and Lerythro-dihydrobiopterin (H2biopterin). Moreover, the ligand-bound forms of the enzyme also showed a decreased resistance to limited tryptic proteolysis. These findings indicate that the binding of H4biopterin 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 H4biopterin which may have significant implications for the physiological regulation of catecholamine biosynthesis in neuroendocrine cells.

Ca2+‐ATPase H+‐ATPase Proton pump Chromaffin granule Adrenal medulla

A subcellular fraction, highly enriched in uncoated vesicles (UCV) with high H+‐ATPase (EC 3.6.1.34) activity, was isolated from the crude microsomal fraction of rat liver homogenates by discontinuous sucrose gradient centrifugation. The UCV fraction, recovered at the interface of sucrose density 1.08 and 1.10 , was shown morphologically to be a mixture of small, smooth‐surfaced univesicular and a few multivesicular structures. A permeable anion (e.g. chloride) was required for internal acidification, indicating an electro‐neutral proton pump. Specific inhibitors of anion transport (pyridoxal 5'‐phosphate and 4‐acetamide‐4'‐isothiocyanostilbene‐2, 2'‐disulfonic acid) totally inhibit proton translocation. The proton pump activity was insensitive to oligomycin, but was completely inhibited by about 5 μM of the tridentate bathophenanthroline chelate of Fe(II). The activity was also inhibited 100% by low concentrations of the protonophore carbonyl cyanide p‐trifluoromethoxyphenylhydrazone, the proton conduction inhibitor N,N'‐dicyclohexyl‐carbodiimide and the ionophore monensin. The UCV fraction contained 2 proteins of M r 50000 (major) and 54000 (minor) which were phosphorylated by an endogenous cyclic nucleotide‐ and Ca2+‐independent protein kinase.

The thiol reagent N‐ethylmaleimide (NEM) completely inhibits the proton pump activity of the H+‐ATPase in chromaffin granule ‘ghosts’ at concentrations which only partly (∼20%) inhibit the Mg2+‐dependent ATP hydrolysis. Half‐maximal inhibition was obtained at ∼13 μM NEM as compared to 18 μM for the classical proton channel inhibitor N,N'‐dicyclohexylcarbodiimide (DCCD), and the apparent stoichiometry of the inhibitors at complete inhibition was NEM:DCCD ⋍ 1 : 2. High concentrations of NEM (⪢100 μM) induce a dissipation of the transmembrane potential generated by MgATP. These findings establish NEM as a valuable proton channel inhibitor in chromaffin granules and explain the rather complex effect of NEM previously reported for catecholamine accumulation in this organelle.