Ko Y H, Bianchet M, Amzel L M, Pedersen P L
Department of Biological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205-2185, USA.
J Biol Chem. 1997 Jul 25;272(30):18875-81. doi: 10.1074/jbc.272.30.18875.
The chemical mechanism by which the F1 moiety of ATP synthase hydrolyzes and synthesizes ATP remains unknown. For this reason, we have carried out studies with orthovanadate (Vi), a phosphate analog which has the potential of "locking" an ATPase, in its transition state by forming a MgADP.Vi complex, and also the potential, in a photochemical reaction resulting in peptide bond cleavage, of identifying an amino acid very near the gamma-phosphate of ATP. Upon incubating purified rat liver F1 with MgADP and Vi for 2 h to promote formation of a MgADP.Vi-F1 complex, the ATPase activity of the enzyme was markedly inhibited in a reversible manner. When the resultant complex was formed in the presence of ultraviolet light inhibition could not be reversed, and SDS-polyacrylamide gel electrophoresis revealed, in addition to the five known subunit bands characteristic of F1 (i.e. alpha, beta, gamma, delta, and epsilon), two new electrophoretic species of 17 and 34 kDa. Western blot and N-terminal sequencing analyses identified both bands as arising from the beta subunit with the site of peptide bond cleavage occurring at alanine 158, a conserved residue within F1-ATPases and the third residue within the nucleotide binding consensus GX4GK(T/S) (P-loop). Quantification of the amount of ADP bound within the MgADP. Vi-F1 complex revealed about 1.0 mol/mol F1, while quantification of the peptide cleavage products revealed that no more than one beta subunit had been cleaved. Consistent with the cleavage reaction involving oxidation of the methyl group of alanine was the finding that [3H] from NaB[3H]4 incorporates into MgADP.Vi-F1 complex following treatment with ultraviolet light. These novel findings provide information about the transition state involved in the hydrolysis of ATP by a single beta subunit within F1-ATPases and implicate alanine 158 as residing very near the gamma-phosphate of ATP during catalysis. When considered with earlier studies on myosin and adenylate kinase, these studies also implicate a special role for the third residue within the GX4GK(T/S) sequence of many other nucleotide-binding proteins.
ATP合酶的F1部分水解和合成ATP的化学机制仍然未知。因此，我们用原钒酸盐（Vi）进行了研究，原钒酸盐是一种磷酸盐类似物，它有可能通过形成MgADP·Vi复合物在其过渡态“锁定”ATP酶，并且在导致肽键断裂的光化学反应中，有可能识别非常靠近ATPγ-磷酸基团的氨基酸。将纯化的大鼠肝脏F1与MgADP和Vi一起孵育2小时以促进MgADP·Vi-F1复合物的形成，该酶的ATP酶活性以可逆方式被显著抑制。当在紫外光存在下形成所得复合物时，抑制作用无法逆转，并且SDS-聚丙烯酰胺凝胶电泳显示，除了F1特有的五条已知亚基带（即α、β、γ、δ和ε）外，还有两条新的17 kDa和34 kDa的电泳条带。蛋白质免疫印迹和N端测序分析确定这两条带均来自β亚基，肽键断裂位点发生在丙氨酸158处，丙氨酸158是F1-ATP酶中的保守残基，也是核苷酸结合共有序列GX4GK(T/S)（P环）中的第三个残基。对MgADP·Vi-F1复合物中结合的ADP量进行定量分析显示，约为1.0 mol/mol F1，而对肽段裂解产物进行定量分析显示，裂解的β亚基不超过一个。与涉及丙氨酸甲基氧化的裂解反应一致的是，在用紫外光处理后，NaB[3H]4中的[3H]掺入到MgADP·Vi-F1复合物中的发现。这些新发现提供了有关F1-ATP酶中单个β亚基水解ATP所涉及的过渡态的信息，并表明丙氨酸158在催化过程中非常靠近ATP的γ-磷酸基团。与早期对肌球蛋白和腺苷酸激酶的研究一起考虑时，这些研究还暗示了许多其他核苷酸结合蛋白的GX4GK(T/S)序列中的第三个残基具有特殊作用。
