EXAFS Studies of the Zinc Binding Sites in Human Porphobilinogen Synthase

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Title: EXAFS Studies of the Zinc Binding Sites in Human Porphobilinogen Synthase
Author: Ginsberg, Nicolle
Advisor: Scarrow, Robert C.
Department: Haverford College. Department of Chemistry
Type: Thesis (B.S.)
Issue Date: 2002
Abstract: Zinc coordination in human porphobilinogen synthase (PBGS) was studied using X-ray Absorption Spectroscopy (XAS) associated with the Zn K edge. Human PBGS is a homo-octamer, which catalyzes the asymmetric condensation of two molecules of 5- aminolaevulinic acid (ALA) into porphobilinogen. PBGS can bind up to 8 zinc ions per octamer, but only needs four of these for full activity. There are two types of zinc binding sites, the ZnA and the ZnB sites. The ZnA site contains only one sulfur ligand and the ZnB site contains predominantly sulfur ligands. To further elucidate the Zn binding sites, human PBGS samples were analyzed using XAS with different concentrations of PBGS in the sample and varying concentrations of Zn bound per octamer. Additional samples were analyzed in the presence and absence of substrate. These were all analyzed in the frozen and the solution form. A column with PBGS immobilized on a CNBr gel was also prepared and buffer with substrate was pumped through this column while it was analyzed using XAS. The XAS spectra for all these samples of human wildtype PBGS were similar and indicate that the Zn binds predominantly to the ZnB site independent of temperature, concentration of protein, or amount of zinc bound per octamer. The presence of substrate/product does not affect the Zn environment, suggesting that substrate/product might bind somewhere else on the active site or substitute for a similar ligand. A sample of the MinusZnB mutant (C122A, C124A, C132A) was analyzed, and this mutant shows binding at the ZnA site. These results suggest that in human PBGS, zinc binds to the ZnB site if it is available and Zn binds to the ZnA site only if the ZnB site is mutated.
Subject: Extended X-ray absorption fine structure
Subject: Zinc compounds -- Synthesis
Subject: Porphobilinogen Synthase
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Ginsberg, Nicolle. "EXAFS Studies of the Zinc Binding Sites in Human Porphobilinogen Synthase". 2002. Available electronically from

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