Event Title

Kinetic Characterization of Porphobilinogen Synthase from Clostridium perfringens

Graduation Year

2013

Location

Atrium, Center for Natural Sciences, Illinois Wesleyan University

Start Date

20-4-2013 9:00 AM

End Date

20-4-2013 10:00 AM

Description

Porphobilinogen synthase (PBGS) catalyzes the condensation of two δ-aminolevulinic acid (ALA) molecules to form porphobilinogen (PBG), which is the first common reaction for the synthesis of tetrapyrroles such as heme and chlorophyll. The kinetic characteristics of PBGS from Clostridium perfringens were previously unknown. An expression plasmid containing the C. perfringens PBGS gene was transformed into E. coli cells, PBGS synthesis was induced using Isopropyl δ-D-1-thiogalactopyranoside (IPGT), and PBGS purification was performed using ion-exchange and hydrophobic interaction chromatography. After purification of PBGS, protein assays were utilized to determine the optimal conditions for PBGS activity. The optimal enzyme concentration and buffer pH were calculated, along with the Vmax and Km values and the influence of metal ions as regulators.

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Apr 20th, 9:00 AM Apr 20th, 10:00 AM

Kinetic Characterization of Porphobilinogen Synthase from Clostridium perfringens

Atrium, Center for Natural Sciences, Illinois Wesleyan University

Porphobilinogen synthase (PBGS) catalyzes the condensation of two δ-aminolevulinic acid (ALA) molecules to form porphobilinogen (PBG), which is the first common reaction for the synthesis of tetrapyrroles such as heme and chlorophyll. The kinetic characteristics of PBGS from Clostridium perfringens were previously unknown. An expression plasmid containing the C. perfringens PBGS gene was transformed into E. coli cells, PBGS synthesis was induced using Isopropyl δ-D-1-thiogalactopyranoside (IPGT), and PBGS purification was performed using ion-exchange and hydrophobic interaction chromatography. After purification of PBGS, protein assays were utilized to determine the optimal conditions for PBGS activity. The optimal enzyme concentration and buffer pH were calculated, along with the Vmax and Km values and the influence of metal ions as regulators.