Kinetic Characterization of Porphobilinogen Synthase from Clostridium perfringens
Submission Type
Event
Expected Graduation Date
2013
Location
Atrium, Center for Natural Sciences, Illinois Wesleyan University
Start Date
4-20-2013 9:00 AM
End Date
4-20-2013 10:00 AM
Disciplines
Biology
Abstract
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.
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.