The porphobilinogen synthases (PBGS's) are ancient proteins, essential to nearly all cellular organisms. They catalyze the first common step in tetrapyrrole biosynthesis (i.e., porphyrin, chlorophyll, vitamin B12, etc.), which is the condensation of two molecules of 5-aminolevulinic acid to form porphobilinogen. To date, only one reaction intermediate is known for this complex reaction. All PBGS's are metalloenzymes and metal ion usage has suffered a unique phylogenetic switch between Zn(II) and Mg(II). Human PBGS is the primary target for lead poisoning and the less frequent of two alleles is reported to predispose humans toward this environmental disease. The proposed studies address various aspects of the PBGS's. The first specific aim uses a novel """"""""designer gene"""""""" approach to produce and purify 100 mg quantities of human and pea PBGS because human PBGS uses Zn(II) and pea PBGS uses Mg(II).
The second aim i s to characterize the PBGS derived from two common human alleles and determine the molecular basis for their apparent Pb(II)-related differences.
This aim i ncludes a thorough kinetic and biochemical characterization of the human proteins, including 13C NMR and collaborative EXAFS and Raman spectroscopies and X-ray crystallography. The Raman data are complementary to the 13C NMR data on active site ligands.
The third aim uses mutagenesis in conjunction with kinetics, metal and substrate binding studies, and magnetic resonance techniques, plus collaborative techniques to probe structure/function relationships in PBGS. The mutations will allow us to characterize the yet unknown intermediates in the PBGS-catalyzed reaction. Some mutations probe individual functions of the two active site Zn(II) of human PBGS and others probe the structural basis for half-sites reactivity in these homo-octameric proteins.
The fourth aim focuses on the intriguing phylogenetic switch between the catalytically essential Zn(II) vs. Mg(II). Studies are directed at pea PBGS and also probe an allosteric Mg(II), absent in mammalian PBGS but present in plant and many microbial PBGS. The sum of the proposed studies is expected to yield new and important information on the mechanism of lead poisoning, on the intermediate in the PBGS catalyzed reaction, on the possible roles of both Zn(II) and Mg(II) in enzyme catalysis, on the structural basis for half sites reactivity, and on the phylogenetic differences between PBGSs. Because PBGS is an essential enzyme with distinct variations between species, we envision that a thorough understanding of the phylogenetic differences will form the basis for the rational design of a new class of organism-specific drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES003654-18
Application #
6382068
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Kirshner, Annette G
Project Start
1991-04-01
Project End
2003-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
18
Fiscal Year
2001
Total Cost
$344,189
Indirect Cost
Name
Institute for Cancer Research
Department
Type
DUNS #
872612445
City
Philadelphia
State
PA
Country
United States
Zip Code
19111
Jaffe, Eileen K (2016) The Remarkable Character of Porphobilinogen Synthase. Acc Chem Res 49:2509-2517
Ramirez, Ursula D; Nikonova, Anna S; Liu, Hanqing et al. (2015) Compounds identified by virtual docking to a tetrameric EGFR extracellular domain can modulate Grb2 internalization. BMC Cancer 15:436
Lentz, Christian S; Halls, Victoria S; Hannam, Jeffrey S et al. (2014) wALADin benzimidazoles differentially modulate the function of porphobilinogen synthase orthologs. J Med Chem 57:2498-510
Lawrence, Sarah H; Selwood, Trevor; Jaffe, Eileen K (2013) Environmental contaminants perturb fragile protein assemblies and inhibit normal protein function. Curr Chem Biol 7:196-206
Jaffe, Eileen K (2013) Impact of quaternary structure dynamics on allosteric drug discovery. Curr Top Med Chem 13:55-63
Jaffe, Eileen K; Lawrence, Sarah H (2012) Allostery and the dynamic oligomerization of porphobilinogen synthase. Arch Biochem Biophys 519:144-53
Selwood, Trevor; Jaffe, Eileen K (2012) Dynamic dissociating homo-oligomers and the control of protein function. Arch Biochem Biophys 519:131-43
Jaffe, Eileen K; Lawrence, Sarah H (2012) The morpheein model of allostery: evaluating proteins as potential morpheeins. Methods Mol Biol 796:217-31
Jaffe, Eileen K; Shanmugam, Dhanasekaran; Gardberg, Anna et al. (2011) Crystal structure of Toxoplasma gondii porphobilinogen synthase: insights on octameric structure and porphobilinogen formation. J Biol Chem 286:15298-307
Lawrence, Sarah H; Selwood, Trevor; Jaffe, Eileen K (2011) Diverse clinical compounds alter the quaternary structure and inhibit the activity of an essential enzyme. ChemMedChem 6:1067-73

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