Kinetic characterization of the reconstituted dicarboxylate carrier from mitochondria: a four-binding-site sequential transport system.
The mitochondrial antiport carriers form a protein family with respect to their structure and function. The kinetic antiport mechanism, being of the sequential type, shows that the dicarboxylate carrier also belongs to this family. This was demonstrated by bireactant initial velocity studies of the purified and reconstituted carrier protein. The transport affinity of the carrier for the internal substrate was largely independent of the external substrate concentration and vice versa, whereas the carrier's apparent Vmax rose with increasing saturation of internal and external binding sites. Thus, the carrier forms a catalytic ternary complex with one internal and one external substrate molecule. As compared to other mitochondrial antiport carriers, however, the situation with the dicarboxylate carrier is more complex. On each membrane side of the protein two separate binding sites exist, one specific for phosphate (or its analogue phenyl phosphate), the other specific for dicarboxylate (or butyl malonate), that can be occupied by the respective substrates without mutual interference. This became evident from the non-competitive interaction of these substrates (or analogues) with the carrier. The two external, but not the two internal binding sites could be saturated simultaneously with phosphate and malate, thereby causing inhibition of transport. All four binding sites must be associated with the same translocation pathway through the carrier protein, since the sequential antiport mechanism held true for the phosphate/malate heteroexchange as well as for the malate/malate or phosphate/phosphate homoexchange.
Indiveri
C
C
Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Italy.
Prezioso
G
G
Dierks
T
T
Krämer
R
R
Palmieri
F
F
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry*; D029382:Dicarboxylic Acid Transporters; D007700:Kinetics; D008293:Malates / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D051381:Rats
D002352:Carrier Proteins; D029382:Dicarboxylic Acid Transporters; D008293:Malates; D010710:Phosphates