Adenosine di- and triphosphate transport in mitochondria. Role of the amidine region for substrate binding and transport.
A variety of base-modified nucleotide analogues was prepared and characterized as their alpha-32P- or U-14C-labeled compounds. Carrier-linked nucleotide binding and carrier-catalyzed exchange across the inner membrane of rat liver mitochondria were measured by using an inhibitor (atractyloside) stop method. Kinetic data of carrier-specific bound analogues were evaluated from Dixon plots and indicate that these analogues are competitive inhibitors for mitochondrial [14C]ADP uptake. Km and Vmax values for carrier-mediated uptake of nucleotide analogues were calculated from Lineweaver-Burk plots. By means of the analogues, a systematic mapping of the essential chemical and steric interactions between the transporter protein and the heterocycle of its substrate in the course of the binding as well as transfer step was achieved. Prerequisites for carrier-specific binding (recognition) are (A) an anti- or syn-positioned beta-glycosyl-linked heterocycle, (B) a nitrogen ring atom in position 7 for syn-structured analogues, and (C) an electron-rich region at the N(1) position, i.e., a permanent dipole moment oriented toward N(1) for anti-structured analogues. Additional requirements for subsequent transport catalysis are (A) a non-fixed anti-positioned base moiety with a beta-glycosyl torsion angle of about -20 degrees, (B) a C(6)-positioned amino group, and (C) an unsubstituted C(2) atom. The complementary binding site at the carrier protein to the N(1)-C(6)(-NH2) amidine region is proposed to be represented by two juxtaposed and invariant bonding points, i.e., an asparagine or glutamine residue.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D007700:Kinetics; D008930:Mitochondria, Liver / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D051381:Rats; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; D009713:Nucleotidyltransferases