Nucleotide binding to uncoupling protein. Mechanism of control by protonation.
Nucleotide binding to the isolated uncoupling protein (UCP) from brown adipose tissue of hamster was studied in detail under equilibrium conditions. Besides microequilibrium dialysis and elution chromatography, a rapid anion-exchange procedure was adapted. From the concentration dependence, the KD and the binding capacity to UCP of ATP, ADP, and GTP and of the ATP analogues 5'-adenylyl imidodiphosphate (AMPPNP) and adenosine 5'-O-(3-thiotriphosphate) were determined. Elucidation of the pH dependence of nucleotide binding was the prime topic. From pH 4.6 to 7.5, the KD varies by almost 2 orders of magnitude, reaching the limits of the equilibrium methods. The pKD of GTP and ATP decreases from 6.3 to 4.3 with increasing pH. For ADP, the pKD varies only from 6.0 to 4.8. The intricate course of the pH dependence shows a "break point" of the pKD around pH 6.3, where the slope (pKD/pH) changes between about -0.2 and -1. Another break point above pH 7.2 produces a pKD/pH = -2 for ATP and GTP only. AMPPNP binding has a lower affinity (pKD about 5.8-4.1) and a pH dependence slope of -1 with no break. The breaks suggest involvement of the last ionization group (pKH approximately equal to 6.7) of the nucleotide phosphate. This agrees with the absence of a break for AMPPNP and with the shift by Mg2+ of the break for ATP to lower pH. The best-fitting model for the pH dependence requires in addition a H+ dissociating group at the binding site of UCP with a pKH approximately equal to 4, dominating the whole pH range. A second group effective above pH 7.0 amplifies the debinding specifically of ATP, not CTP or ADP. Further, the model implies binding of both NTP4- and the protonated NTPH3- or NDP3- and NDPH2- forms, however, with different affinities. On this basis, the relation between the measured overall KD and the intrinsic KD's of both nucleotide forms and the various H+ dissociation constants is derived, and the corresponding pKD/pH curves are calculated. A good fit with the data is obtained with a pKH = 3.8 for the UCP center and a pKH = 6.8 for nucleotides and with affinity ratios of 50 for NTP4-/NTPH3- and 100 for NDP3-/NDPH2-. The binding of the protonated nucleotide NTPH3- is seen only at a low pH, but with the analogue AMPPNPH3- with pKH = 7.6 it dominates the whole pH range to pH 7.2 with corresponding low affinity.(ABSTRACT TRUNCATED AT 400 WORDS)
Klingenberg
M
M
Institut für Physikalische Biochemie, Universität München, FRG.
D000227:Adenine Nucleotides / Q000378:metabolism; D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins; D006224:Cricetinae; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D011485:Protein Binding; D014475:Uncoupling Agents / Q000378:metabolism*; D000071256:Uncoupling Protein 1
D000227:Adenine Nucleotides; D002352:Carrier Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D014475:Uncoupling Agents; D000071256:Uncoupling Protein 1; D006160:Guanosine Triphosphate; D008274:Magnesium