Seto-Young D, Na S, Monk B C, Haber J E, Perlin D S
Public Health Research Institute, New York, New York 10016.
J Biol Chem. 1994 Sep 30;269(39):23988-95.
Transmembrane segments 1 and 2 of the yeast plasma membrane H(+)-ATPase are believed to form a helical hairpin structure that is joined by a short extracytoplasmic loop. The hairpin head region (Ala135-Phe144) was probed using site-directed mutagenesis. Scanning alanine mutagenesis produced functional H(+)-ATPase at all positions except Leu138, Asp143, and Phe144. D140A and V142A gave strong hygromycin B resistance and low pH sensitivity suggesting a major kinetic defect in these mutant enzymes. Other amino acid substitutions, such as L138Y, were highly perturbing, while mutations S139E and D140E produced minor effects on phenotype. Small uncharged residues Gly and Ala, which were inserted between Leu138 and Ser139 to examine the importance of loop length on H(+)-ATPase function, were well tolerated, while the insertion of a polar Ser residue was highly perturbing. Other additions were not tolerated by the enzyme. These results suggest that the turn region has limited structural flexibility. The conserved Phe144 residue could be changed to Trp with a minor effect on phenotype. However, neither Tyr, Arg, nor small hydrophobic residues could substitute, suggesting that this region is closely packed and hydrophobic. ATP hydrolysis measurements showed that Vmax was significantly reduced in nearly all mutant enzymes, except D140E; whereas, Km values were nearly normal. Vanadate-sensitivity and pH profiles for ATP hydrolysis were nearly normal for all mutant enzymes except insertion mutant S138+. Mutants with extreme phenotypes (S138+, Tyr138) showed significantly altered medium acidification profiles. These results support the notion that the hairpin head region linking transmembrane segments 1 and 2 forms a tightly packed conformationally sensitive domain that is coupled to the catalytic ATP hydrolysis domain.
酵母质膜H(+)-ATP酶的跨膜片段1和2被认为形成了一个螺旋发夹结构，该结构由一个短的胞外环连接。使用定点诱变技术对发夹头部区域（Ala135 - Phe144）进行了探测。扫描丙氨酸诱变在除Leu138、Asp143和Phe144之外的所有位置都产生了功能性的H(+)-ATP酶。D140A和V142A产生了很强的潮霉素B抗性和低pH敏感性，表明这些突变酶存在主要的动力学缺陷。其他氨基酸取代，如L138Y，具有高度干扰性，而突变S139E和D140E对表型产生了较小影响。在Leu138和Ser139之间插入小的不带电荷的残基Gly和Ala以研究环长度对H(+)-ATP酶功能的重要性，这些插入很好地被耐受，而插入一个极性Ser残基则具有高度干扰性。酶不能耐受其他添加物。这些结果表明转折区域的结构灵活性有限。保守的Phe144残基可以被改变为Trp，对表型的影响较小。然而，Tyr、Arg或小的疏水残基都不能替代，这表明该区域紧密堆积且具有疏水性。ATP水解测量表明，除D140E外，几乎所有突变酶的Vmax都显著降低；而Km值几乎正常。除插入突变体S138+外，所有突变酶的钒酸盐敏感性和ATP水解的pH曲线几乎正常。具有极端表型的突变体（S138+、Tyr138）显示出介质酸化曲线有显著改变。这些结果支持这样一种观点，即连接跨膜片段1和2的发夹头部区域形成了一个紧密堆积的构象敏感结构域，该结构域与催化ATP水解结构域相偶联。
