Porphobilinogen synthase (PBGS) is an ancient protein, essential to nearly all-cellular organisms. PBGS catalyzes the first common step in tetrapyrrole biosynthesis (i.e., porphyrin, chlorophyll, vitamin B12), which is the condensation of two molecules of 5-aminolevulinic acid to form porphobilinogen. Despite multiple crystal structures, the order and identity of the catalytic steps remains unclear. The PBGS family consists of metalloproteins among which the utilization of metal ions has a unique phylogenetic variation between Zn2+, Mg2+, and K+. Three hypotheses drive the proposed studies. First, we propose that there are mechanistic differences between the PBGS that use Zn2+ and those that do not. Second, we propose that there is a physiological significance to our newly observed hexameric form of PBGS. All previously deposited crystal structures show an octamer. Third, we propose that a subunit-to-subunit communication outside the active site is the structural basis for the half-site reactivity evident in PBGS. Four interrelated Aims address these hypotheses. ? ? Aim 1 is directed at elucidating the human Zn2+-PBGS catalyzed reaction mechanism. One novel approach uses a variant designed to process two different substrates, instead of two ALA molecules. We propose to use kinetic techniques, isotope effect determinations, 13C and 15N NMR, and other collaborative approaches. ? Aim 2 focuses on the newly discovered hexameric form of PBGS. A hexamer-octamer transition is proposed to be the structural basis for allosteric activation by Mg 2+ for some PBGS. We will also probe for a relationship between altemate quaternary forms of the protein and an allelic variation reported to predispose some humans toward the environmental disease of lead poisoning. ? Aim 3 describes investigation of the Mg2+ requiring PBGS where we focus on mechanism, structure, and the quaternary structure equilibria. ? Aim 4 focuses on Drosophila melanogaster PBGS, which is a superior system for the study of the Zn 2+ utilizing PBGS and an excellent model for probing the subunit-to-subunit communication required for half-site reactivity. In support of these aims are collaborations to probe the structure and function of PBGS using X-ray crystallography, Raman spectroscopy, and analytical ultracentrifugation. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES003654-24
Application #
7367886
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Maull, Elizabeth A
Project Start
1991-04-01
Project End
2010-09-22
Budget Start
2008-03-01
Budget End
2010-09-22
Support Year
24
Fiscal Year
2008
Total Cost
$372,964
Indirect Cost
Name
Research Institute of Fox Chase Cancer Center
Department
Type
DUNS #
064367329
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

Showing the most recent 10 out of 49 publications