Probing the oligomeric assemblies of pea porphobilinogen synthase by analytical ultracentrifugation.
The enzyme porphobilinogen synthase (PBGS) can exist in different nonadditive homooligomeric 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 [Breinig, S., et al. (2003) Nat. Struct. Biol. 10, 757-763]. 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 documented under some conditions, both assemblies are very stable (under AU conditions) in the time frame of our ultracentrifuge experiments.
Kokona
Bashkim
B
Department of Biology, Haverford College, 370 Lancaster Avenue, Haverford, Pennsylvania 19041, USA.
Rigotti
Daniel J
DJ
Wasson
Andrew S
AS
Lawrence
Sarah H
SH
Jaffe
Eileen K
EK
Fairman
Robert
R
D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D015636:Magnesium Chloride / Q000494:pharmacology; D008958:Models, Molecular; D000623:Porphobilinogen Synthase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D014461:Ultracentrifugation / Q000379:methods*
D015636:Magnesium Chloride; D000623:Porphobilinogen Synthase
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