Superstoichiometric binding of L-Phe to phenylalanine hydroxylase from Caenorhabditis elegans: evolutionary implications

Phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of L-Phe to L-Tyr. Dysfunctional PAH results in phenylketonuria and mammalian PAH is therefore highly regulated and displays positive cooperativity for L-Phe (Hill coefficient (h) = 2). L-Phe does not bind to the regulatory ACT domain in full-length tetrameric human PAH and cooperativity is elicited by homotropic binding to the catalytic site (Thórólfsson et al. in Biochemistry 41:7573–7585, 2002). PAH from Caenorhabditis elegans (cePAH) is devoid of cooperativity for L-Phe (h = 0.9), and, as shown in this work, structural analysis reveal an additional L-Phe binding site at the regulatory domain of full-length cePAH. This site involves the GA(S)L/ISRP motifs, which are also found in ACT domains of other L-Phe binding proteins, such as prephenate dehydratase. Isothermal titration calorimetry further demonstrated 2 binding sites per subunit for cePAH versus ~1 for hPAH. Steric occlusion of the regulatory site, notably by residues Lys215/Tyr216 from the adjacent catalytic domain, appears to hinder regulatory binding in full-length hPAH. Accordingly, the humanized mutant Q215K/N216Y of cePAH binds ~1.4 L-Phe/subunit. This mutant also displays high catalytic activity and certain positive cooperativity for L-Phe (h = 1.4). Our results support that the acquisition of positive cooperativity in mammalian forms of PAH is accompanied by a closure of the regulatory L-Phe binding site. Concomitantly, the function of the regulatory ACT domain appears to be adapted from amino acid binding to serving the communication of conformational changes among catalytic subunits.     

Tissue Distribution of 14C-Diflubenzuron in Atlantic Salmon (Salmo salar)

Diflubenzuron is a potent inhibitor of chitin synthesis, with potential use against salmon lice infestations. The absorption, distribution and elimination of the substance in Atlantic salmon was examined after a single, oral dose of 75 mg/kg body weight. The kinetic properties were studied by whole-body autoradiography, liquid scintillation counting and thin layer chromatography, using a 14C-labelled isotope of the substance. The drug was poorly absorbed from the intestine, but reached a concentration of more than 4 µg/g in the mucus layer of the skin 2 days after administration. If maintained for several days, this concentration is probably sufficient to control all moulting stages of sea lice in Atlantic salmon. The main route of excretion was via the bile.