Mechanism of inhibition of the dicarboxylate carrier of mitochondria by thiol reagents.
The nature of the inhibition of the dicarboxylate carrier by compounds reacting with SH groups has been investigated. (1) Mersalyl and p-hydroxymercuribenzoate increase the Km without changing the V of malonate/Pi exchange, when they are added simultaneously with the dicarboxylate. If, on the other hand, the mitochondria are preincubated with SH reagents prior to the addition of malonate, the mersalyl inhibition of malonate/Pi exchange becomes predominantly non-competitive with respect to malonate. (2) In the case of Pi/Pi exchange, catalyzed by the dicarboxylate carrier, the mersalyl inhibition is competitive with respect to Pi (as indicated by Lineweaver-Burk plots), even when mersalyl is added before the substrate. Dixon plots of the rate of Pi uptake against mersalyl concentration are, however, non-linear, suggesting that the inhibition is partially competitive. (3) Dicarboxylates and dicarboxylate analogous protect against SH reagent inhibition of both dicarboxylate and Pi uptake via the dicarboxylate carrier. The protectors are effective when added before, or together with the SH reagents, but do not reverse the inhibition once it has been established. Protection by substrate analogues progressively decreases, as the time of incubation with the SH reagent increases. (4) The presence of Pi does not protect against the SH reagent inhibition of the Pi uptake. (5) The rate of SH reagent inhibition of the dicarboxylate carrier is competively inhibited by dicarboxylates. (6) It is concluded that SH reagents bind at or near the dicarboxylate specific binding site and distant from the Pi binding site. As a result of this reaction these inhibitors prevent dicarboxylate binding directly and decrease the affinity for Pi by an indirect conformational change.
Palmieri
F
F
Department of Biochemistry and Centro di Studio sui Mitocondri e Metabolismo Energetico del CNR, University of Bari, Italy.
Passarella
S
S
Stipani
I
I
Quagliariello
E
E
D000818:Animals; D029382:Dicarboxylic Acid Transporters / Q000037:antagonists & inhibitors / Q000378:metabolism; D003998:Dicarboxylic Acids / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D008314:Malonates / Q000378:metabolism; D008634:Mersalyl / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism*; D051381:Rats; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000737:chemistry / Q000378:metabolism*
D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids; D004791:Enzyme Inhibitors; D008314:Malonates; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D008634:Mersalyl; C030290:malonic acid