title: A structural basis for half-of-the-sites metal binding revealed in Drosophila melanogaster porphobilinogen synthase.
abstract: Porphobilinogen synthase (PBGS) proteins fall into several distinct groups with different metal ion requirements. Drosophila melanogaster porphobilinogen synthase (DmPBGS) is the first non-mammalian metazoan PBGS to be characterized. The sequence shows the determinants for two zinc binding sites known to be present in both mammalian and yeast PBGS, proteins that differ in the exhibition of half-of-the-sites metal binding. The pH-dependent activity of DmPBGS is uniquely affected by zinc. A tight binding catalytic zinc binds at 0.5/subunit with a Kd well below microm. A second inhibitory zinc exhibits a Kd of approximately 5 microm and appears to bind at a stoichiometry of 1/subunit. A molecular model of DmPBGS suggests that the inhibitory zinc is located at a subunit interface using Cys-219 and His-10 as ligands. Zinc binding to this previously unknown inhibitory site is proposed to inhibit opening of the active site lid. As predicted, the DmPBGS mutant H10F is active but is not inhibited by zinc. H10F binds a catalytic zinc at 0.5/subunit and binds a second nonessential and noninhibitory zinc at 0.5/subunit. This result reveals a structural basis for half-of-the-sites metal binding that is consistent with a reciprocating motion model for function of oligomeric PBGS.
authors.affiliation: Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111-2497, USA.
authors.forename: Lenka
authors.initials: L
authors.lastname: Kundrat
authors.forename: Jacob
authors.initials: J
authors.lastname: Martins
authors.forename: Linda
authors.initials: L
authors.lastname: Stith
authors.forename: Roland L
authors.initials: RL
authors.lastname: Dunbrack
authors.forename: Eileen K
authors.initials: EK
authors.lastname: Jaffe
mesh_terms: D000818:Animals; D004331:Drosophila melanogaster / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D016296:Mutagenesis; D000623:Porphobilinogen Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013045:Species Specificity; D013329:Structure-Activity Relationship; D015032:Zinc / Q000378:metabolism*
chemical_list: D000623:Porphobilinogen Synthase; D015032:Zinc