Echtay K S, Bienengraeber M, Winkler E, Klingenberg M
Institute of Physical Biochemistry, University of Munich, Schillerstrasse 44, 80336 Munich, Germany.
J Biol Chem. 1998 Sep 18;273(38):24368-74. doi: 10.1074/jbc.273.38.24368.
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.
棕色脂肪组织解偶联蛋白（UCP - 1）与核苷酸的结合受pH调节。有人提出，核苷酸磷酸部分的结合口袋受羧基（pK约为4.5）质子化的控制，核苷二磷酸（NDP）和核苷三磷酸（NTP）均是如此（该羧基被确定为Glu - 190），而对于NTP，仅有一个组氨酸（pK约为7.2）参与调控。在此，我们确定His - 214是仅对NTP结合具有特异性的pH传感器。在仓鼠重组的UCP - 1中，焦碳酸二乙酯（DEPC）减少NTP的结合，但不影响NDP的结合。它还能阻止NTP对H⁺运输的抑制，但不能阻止NDP的这种抑制作用。在酿酒酵母中表达的仓鼠UCP - 1突变为H214N后，对NTP（GTP）的结合亲和力仅发生适度变化，但在H214W中，由于取代基体积更大，亲和力下降了10倍，而对NDP（ADP）的亲和力基本不变。野生型中NTP结合亲和力随pH的急剧下降（从pH 6.0至7.5）在突变体中变得平缓得多。此外，这些突变体中结合和解离速率对pH的依赖性也降低了。H⁺和Cl⁻的运输不受影响。因此，His - 214仅参与核苷酸结合，而如先前所示，His - 145和His - 147仅参与H⁺运输。这些结果证实了之前的推测，即除了一个羧基控制NTP和NDP的结合外，还有一个组氨酸调节NTP的结合。有人提出，His - 2突入γ - 磷酸的结合口袋从而抑制NTP结合，并且His214H⁺被一个背景 -CO₂⁻基团拉回，为γ - 磷酸让路。
