Abstract
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.
Highlights
From the Department of Physiology, The Johns Hopkins UniversitySchool of Medicine, Baltimore, Maryland 21205 and the SNational Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa 50010
Purified OxlT has a turnover number of at of equilibrium between liganded and unliganded spe- least 500/s (3), some 10-100-fold greater than most other cies, allowed calculation of substrate dissociation cons-econdary transporters, and this high catalytic effistants for oxalate (18 2 3 p ~ ) m, alonate (1.2 & 0.2 ciency is of special interest, itmakes conventional techniques mM),and formate (3.1 2 0.6 mM)
It has been difficult to revealed that substrate binding energy contributed at directly assess the role of formate, since its effects on OxlTleast 3.6 kcal/mol to stabilizationof solubilized OxlT. mediated exchanges maybe secondary tothe changes of pAghrisiaraceodnvcraiioesrzc.rweahdctiiitdnoltyeognrlyidnaa,unvwproapeillnlvyisgczeuaimdgntgigeioenmnsptbdirrrntoaihtvnteaiheintnetg-rsssuaputnburbssdosptttyroreaariontttfe.esTmtbihrnieuinsmtcedntrbiueanrwrcgaetmnciaoaeerpannrey-s-oerrg- imirynneectTetaoohrnnnetsaatdfiltotdeuprcrmteHhisoniscncotaalho(uc4egisd-sey6e.d)oibsftoysumdpeeeasmvswsebielvoraehpnamneveeowpverexommpteeleoitnnhittoesdodosflrutteohbceeialninhztaiaglatyhidozlvyenanspaucenberd-sstrate-protein interactions inOxlT
Summary
Vol 267, Noof . 15, Issue May 25, pp. 1P0r5i3n1te-1d05in36U,1.S99.A2. Measurement of the Substrate Dissociation Constant of a Solubilized Membrane Carrier. The concentration dependence of substrate protections could be interpreted by a simple model to estimate the dissociation constant ( K D ) of the carrier-substrate complex; for OxlT this has been useful in quantitating parameters less confidently estimated by standard kinetic tests. These experiments provided direct evidence of an interaction between OxlT and formate, reinforcing the idea that formate plays a role in a biological setting.
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