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.
Flydal
Marte I
MI
Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009, Bergen, Norway.
Mohn
Tonje C
TC
Pey
Angel L
AL
Siltberg-Liberles
Jessica
J
Teigen
Knut
K
Martinez
Aurora
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D055162:Biocatalysis; D017173:Caenorhabditis elegans / Q000201:enzymology*; D002151:Calorimetry; D002942:Circular Dichroism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry*; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013237:Stereoisomerism
D010649:Phenylalanine; D010651:Phenylalanine Hydroxylase