Seto-Young D, Monk B, Mason A B, Perlin D S
Public Health Research Institute, New York, NY 10016, USA.
Biochim Biophys Acta. 1997 Jun 12;1326(2):249-56. doi: 10.1016/s0005-2736(97)00028-x.
The plasma membrane H(+)-ATPase is a promising new antifungal target that is readily probed with the sulfhydryl-reactive reagent omeprazole. Inhibition of the H(+)-ATPase by omeprazole is closely linked to cell killing, and it has been suggested that enzyme inhibition may result from a covalent interaction within the first two transmembrane segments (M1 and M2) (Monk et al. (1995) Biochim. Biophys. Acta 1239, 81-90). In this study, the molecular nature of this interaction was examined by screening a series of 26 well-characterized pma1 mutations residing in the first two transmembrane segments of the H(+)-ATPase from Saccharomyces cerevisiae. Only two pma1 mutants, A135G and G158D,G156C, were found to significantly decrease the sensitivity of cells for omeprazole. In contrast, enhanced sensitivity was observed at a number of positions, with D140C(A) and M128C producing the most significant increases in sensitivity. The introduction of cysteine at various locations within this region only marginally affected omeprazole sensitivity, suggesting that this region was not a direct site of covalent modification. Rather, its conformation influences omeprazole binding at some other locus. In order to determine the sidedness of the omeprazole interaction, a novel in vitro assay system was exploited that utilized liposomes co-reconstituted with the H(+)-ATPase and the light-driven proton pump bacteriorhodopsin. Omeprazole was found to completely inhibit proton transport by the H(+)-ATPase at 50 microM in this system. An asymmetrically-distributed chemical trap system involving glutathione was used to demonstrate that this inhibition appears localized to the extracellular portion of the enzyme. This work indicates that omeprazole can inhibit the H(+)-ATPase from its extracellular face, and this inhibition is influenced by changes in the M1, M2 region of the protein.
质膜H(+)-ATP酶是一个很有前景的新型抗真菌靶点，可用巯基反应试剂奥美拉唑轻易探测。奥美拉唑对H(+)-ATP酶的抑制作用与细胞杀伤密切相关，有人提出酶抑制可能是由于在前两个跨膜片段（M1和M2）内发生了共价相互作用（Monk等人，(1995)《生物化学与生物物理学报》1239, 81 - 90）。在本研究中，通过筛选酿酒酵母H(+)-ATP酶前两个跨膜片段中一系列26个特征明确的pma1突变体，研究了这种相互作用的分子本质。仅发现两个pma1突变体A135G和G158D、G156C能显著降低细胞对奥美拉唑的敏感性。相反，在多个位点观察到敏感性增强，其中D140C(A)和M128C导致的敏感性增加最为显著。在该区域内不同位置引入半胱氨酸仅对奥美拉唑敏感性有轻微影响，表明该区域不是共价修饰的直接位点。相反，其构象在其他某个位点影响奥美拉唑的结合。为了确定奥美拉唑相互作用的方向，利用了一种新型体外测定系统，该系统使用了与H(+)-ATP酶和光驱动质子泵细菌视紫红质共重构脂质体。在该系统中发现，奥美拉唑在50微摩尔浓度时能完全抑制H(+)-ATP酶的质子转运。使用涉及谷胱甘肽的不对称分布化学捕获系统证明这种抑制似乎局限于酶的细胞外部分。这项工作表明奥美拉唑可从其细胞外表面抑制H(+)-ATP酶，并且这种抑制受到蛋白质M1、M2区域变化的影响。
