title: Investigations on the metal switch region of human porphobilinogen synthase.
abstract: Porphobilinogen synthase (PBGS) is an ancient and highly conserved protein that functions in the first common step in tetrapyrrole biosynthesis. The PBGS protein sequence contains a unique metal switch region that has been postulated to dictate an exclusive catalytic use of either zinc or magnesium, and perhaps also potassium. In some PBGS, the cysteines of the metal switch sequence DXCXCX(Y/F)X(3)G(H/Q)CG have been demonstrated to bind a catalytic zinc, and in other PBGS, the aspartic acid residues of the metal switch sequence DXALDX(Y/F)X(3)G(H/Q)DG have been postulated to bind a catalytically essential magnesium and/or potassium. The current work describes chimeric proteins that contain the aspartate-rich sequences of pea PBGS and Pseudomonas aeruginosa PBGS in place of the naturally occurring cysteine-rich sequence of human PBGS. The resultant chimeric PBGS proteins, peainhuman PBGS and psuinhuman PBGS, are substantially activated by both magnesium and potassium, but not by zinc. The specific activities of the chimeras are significantly lower than human PBGS. Detailed kinetic and inhibition data are presented for both chimeric proteins and are discussed in terms of this unique phylogenetic variation in metal ion usage. The identity of a basic residue, which is Arg221 in human PBGS, strictly correlates with the presence or absence of the cysteine-rich sequence. Those PBGS with the aspartate-rich metal switch sequence contain Lys in the analogous position. The R221K mutation was inserted into wild type and chimeric human PBGS and found to further reduce the activity of both, illustrating the subtle nature of the role of this residue.
authors.affiliation: Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA. EK_Jaffe@fccc.edu
authors.forename: Eileen K
authors.initials: EK
authors.lastname: Jaffe
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D017124:Conserved Sequence; D003652:Decanoic Acids / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000623:Porphobilinogen Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
chemical_list: C434703:4,7-dioxosebacic acid; C434705:4-oxosebacic acid; D003652:Decanoic Acids; D008670:Metals; D011993:Recombinant Fusion Proteins; D000623:Porphobilinogen Synthase; D008274:Magnesium; D015032:Zinc; D008239:Lysine; D011188:Potassium