Probing the Oligomeric State of Pea Porphobilinogen Synthase by Analytical Ultracentrifugation

Bashkim Kokono
Daniel J. Rigotti
Andrew S. Wasson '02, Class of 2002 Haverford College
Sarah H. Lawrence
Robert Fairman, Haverford College


The enzyme porphobilinogen synthase (PBGS) can exist in different non-additive homo-oligomeric assemblies and, under appropriate conditions, the distribution of these assemblies can respond to ligands such as metals or substrate. PBGS from most organisms was believed to be octameric until work on a rare allele of human PBGS revealed an alternate hexameric assembly, which is also available to the wild type enzyme at elevated pH. Herein, we establish that the distribution of pea PBGS quaternary structures also contains octamers and hexamers, using both sedimentation velocity and sedimentation equilibrium experiments. We report results in which the octamer dominates under purification conditions and discuss conditions that influence the octamer:hexamer ratio. As predicted by PBGS crystal structures from related organisms, in the absence of magnesium, the octameric assembly is significantly destabilized and the oligomeric distribution is dominated largely by the hexameric assembly. Although the PBGS hexamer-to-octamer oligomeric rearrangement is well document under some conditions, both assemblies are very stable (under AUC conditions) in the timeframe of our ultracentrifuge experiments.