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

Flydal, MI, Mohn, TC, Pey, AL et al. (2010). Superstoichiometric binding of L-Phe to phenylalanine hydroxylase from Caenorhabditis elegans: Evolutionary implications . 39(5), 1463-1475. 10.1007/s00726-010-0611-6

cited authors

  • Flydal, MI; Mohn, TC; Pey, AL; Siltberg-Liberles, J; Teigen, K; Martinez, A

authors

abstract

  • 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. © 2010 Springer-Verlag.

publication date

  • November 1, 2010

Digital Object Identifier (DOI)

start page

  • 1463

end page

  • 1475

volume

  • 39

issue

  • 5