Valiakhmetov Airat, Perlin David S
Public Health Research Institute, Newark, New Jersey 07103, USA.
J Biol Chem. 2003 Feb 21;278(8):6330-6. doi: 10.1074/jbc.M208927200. Epub 2002 Dec 11.
The molecular architecture of the yeast plasma membrane H(+)-ATPase phosphorylation region was explored by Fe(2+)-catalyzed cleavage. An ATP-Mg(2+).Fe(2+) complex was found to act as an affinity cleavage reagent in the presence of dithiothreitol/H(2)O(2). Selective enzyme cleavage required bound adenine nucleotide, either ATP or ADP, in the presence of Mg(2+). The fragment profile included a predominant N-terminal 61-kDa fragment, a minor 37-kDa fragment, and three prominent C-terminal fragments of 39, 36, and 30 kDa. The 61-kDa N-terminal and 39-kDa C-terminal fragments were predicted to originate from cleavage within the conserved MLT(558)GDAVG sequence. The 37-kDa fragment was consistent with cleavage within the S4/M4 sequence PVGLPA(340)V, while the 30-kDa and 36-kDa C-terminal fragments appeared to originate from cleavage in or around sequences D(646)TGIAVE and DMPGS(595)ELADF, respectively. The latter are spatially close to the highly conserved motif GD(634)GVND(638)APSL and conserved residues Thr(558) and Lys(615), which have been implicated in coordinating Mg(2+) and ATP. Overall, these results demonstrate that Fe(2+) associated with ATP and Mg(2+) acts as an affinity cleavage agent of the H(+)-ATPase with backbone cleavage occurring in conserved regions known to coordinate metal-nucleotide complexes. This study provides support for a three-dimensional organization of the phosphorylation region of the yeast plasma membrane H(+)-ATPase that is consistent with, but not identical to, typical P-type enzymes.
通过Fe(2+)催化裂解研究了酵母质膜H(+)-ATP酶磷酸化区域的分子结构。发现ATP-Mg(2+).Fe(2+)复合物在二硫苏糖醇/H2O2存在下可作为亲和裂解试剂。在Mg(2+)存在下，选择性酶裂解需要结合的腺嘌呤核苷酸，即ATP或ADP。片段图谱包括一个主要的N端61 kDa片段、一个较小的37 kDa片段和三个突出的C端片段，分别为39、36和30 kDa。预测61 kDa的N端片段和39 kDa的C端片段源自保守的MLT(558)GDAVG序列内的裂解。37 kDa片段与S4/M4序列PVGLPA(340)V内的裂解一致，而30 kDa和36 kDa的C端片段似乎分别源自D(646)TGIAVE序列内或其附近以及DMPGS(595)ELADF序列内的裂解。后者在空间上靠近高度保守的基序GD(634)GVND(638)APSL以及保守残基Thr(558)和Lys(615)，它们参与协调Mg(2+)和ATP。总体而言，这些结果表明与ATP和Mg(2+)相关的Fe(2+)作为H(+)-ATP酶的亲和裂解剂，主链裂解发生在已知可协调金属-核苷酸复合物的保守区域。本研究为酵母质膜H(+)-ATP酶磷酸化区域的三维组织提供了支持，该组织与典型的P型酶一致但不完全相同。
