Measurement of the substrate dissociation constant of a solubilized membrane carrier. Substrate stabilization of OxlT, the anion exchange protein of Oxalobacter formigenes.
OxlT, a secondary carrier found in Oxalobacter formigenes, mediates the exchange of divalent oxalate and monovalent formate. Because OxlT has an unusually high turnover number (greater than or equal to 1000/s), and because formate, one its substrates, shows high passive membrane permeability as formic acid, it has been difficult to obtain information on protein-substrate interactions using traditional methods in membrane biology. For this reason, we devised a new way to measure substrate dissociation constants. Detergent-solubilized material was exposed to inactivating temperatures in the absence or presence of OxlT substrates, and periodic reconstitution was used to monitor the kinetics of thermal decay. The data were consistent with a simple scheme in which only unliganded OxlT was temperature-sensitive; this premise, along with the assumption of equilibrium between liganded and unliganded species, allowed calculation of substrate dissociation constants for oxalate (18 +/- 3 microM), malonate (1.2 +/- 0.2 mM), and formate (3.1 +/- 0.6 mM). Further analysis revealed that substrate binding energy contributed at least 3.5 kcal/mol to stabilization of solubilized OxlT. Accordingly, we suggest that substrate binding energy is directly involved in driving protein structure reorganization during membrane transport. This new approach to analyzing protein-substrate interactions may have wider application in the study of membrane carriers.
Maloney
P C
PC
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Anantharam
V
V
Allison
M J
MJ
D000838:Anions; D001692:Biological Transport; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005561:Formates / Q000378:metabolism; D006089:Gram-Negative Anaerobic Bacteria / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008314:Malonates / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D010070:Oxalates / Q000378:metabolism; D013379:Substrate Specificity; D014634:Valinomycin / Q000494:pharmacology
D000838:Anions; D005561:Formates; D008314:Malonates; D008565:Membrane Proteins; D010070:Oxalates; D014634:Valinomycin; C030290:malonic acid