In the uncoupling protein (UCP-1) His-214 is involved in the regulation of purine nucleoside triphosphate but not diphosphate binding. J Biol Chem 1998 Sep 18;273(38):24368-74
Date
09/12/1998Pubmed ID
9733725DOI
10.1074/jbc.273.38.24368Scopus ID
2-s2.0-0032544693 (requires institutional sign-in at Scopus site) 34 CitationsAbstract
The nucleotide binding to uncoupling protein (UCP-1) of brown adipose tissue is regulated by pH. The binding pocket of the nucleotide phosphate moiety has been proposed to be controlled by the protonization of a carboxyl group (pK approximately 4.5) for both nucleoside diphosphates (NDP) and nucleoside triphosphates (NTP) (identified as Glu-190) and of a histidine (pK approximately 7. 2) for NTP only. Here we identify His-214 as a pH sensor specific for NTP binding only. In reconstituted UCP-1 from hamster, DEPC diminishes binding of NTP but not of NDP. It also prevents inhibition of H+ transport by NTP but not by NDP. Hamster UCP-1 expressed in Saccharomyces cerevisiae was mutated to H214N resulting in only moderate change of the binding affinity for NTP (GTP) but a 10-fold affinity decrease with the bulkier substituent in H214W, whereas the affinity for NDP (ADP) was largely unchanged. The steep decrease with pH of the binding affinity for NTP in wild type (from pH 6.0 to 7.5) was much flatter in the mutants. Also, the pH dependence of binding and dissociation rates was diminished in these mutants. The transport of H+ and Cl- was not affected. Thus, His-214 is only involved in nucleotide binding, whereas, as previously shown, His-145 and His-147 are involved only in H+ transport. The results validate the earlier proposal of a histidine regulating the NTP binding in addition to a carboxyl group controlling both NTP and NDP binding. It is proposed that His-214 protrudes into the binding pocket for the gamma-phosphate thus inhibiting NTP binding and that His214H+ is retracted by a background -CO2- group to give way for the gamma-phosphate.
Author List
Echtay KS, Bienengraeber M, Winkler E, Klingenberg MMESH terms used to index this publication - Major topics in bold
Adipose Tissue, BrownAmino Acid Substitution
Animals
Binding Sites
Carrier Proteins
Cloning, Molecular
Cricetinae
Diethyl Pyrocarbonate
Guanosine Diphosphate
Guanosine Triphosphate
Histidine
Hydrogen-Ion Concentration
Ion Channels
Kinetics
Membrane Proteins
Mitochondria
Mitochondrial Proteins
Models, Molecular
Mutagenesis, Site-Directed
Point Mutation
Protein Structure, Secondary
Protons
Recombinant Proteins
Substrate Specificity
Uncoupling Protein 1
