質子傳送焦磷酸水解酶(簡稱H+-PPase, EC 3.6.1.1) 是一種可維持生物體內酸鹼恆定的重要酵素，其利用水解焦磷酸產生能量，來驅動質子通過細胞膜藉以調整並維持膜內外氫離子的濃度梯度，以協助其他重要離子及代謝物的穿膜運輸。在植物上此酵素已有些研究，本篇論文乃選擇了破傷風桿菌(Clostridium tetani)中的質子傳送焦磷酸水解酶作為研究對象，繼續探討在細菌中此種酵素的特性。過去的研究指出，酪胺酸在質子傳送過程中扮演了重要的角色，其官能基中的羥基可用於接受及釋放氫離子。因此，我們利用定點突變法以丙胺酸個別取代破傷風桿菌質子傳送焦磷酸水解酶中19個酪胺酸，並檢測其水解焦磷酸活性、質子傳送能力及耦合效率，發現在Y175、Y226、Y392、Y414及Y471突變後皆會造成酵素活性明顯下降，且此五點在許多物種中皆為高度保留，觀察立體結構也發現此五點剛好圍繞於質子通道旁，藉此我們推測這五個位置可能參與了破傷風桿菌質子傳送焦磷酸水解酶催化活性。接著，我們將此五點突變成其他胺基酸，發現在Y414這個位置以絲胺酸及蘇胺酸取代後，其酵素活性可大約回復至野生株的75%，可見羥基在此位置對於酵素的重要性。進一步地，我們以不同離子處理，來觀察對質子傳送焦磷酸水解酶的影響，更發現Y414位置可能與鈉離子的結合有相關聯。根據上述的實驗結果，對於酪胺酸在破傷風桿菌質子傳送焦磷酸水解酶中所扮演的角色，獲得更深入的了解。

Proton-translocating pyrophosphatase (H+-PPase, EC 3.6.1.1) is a crucial enzyme which sustains pH homeostasis of organisms. This enzyme generates and maintains the proton gradient across the vacuolar membrane by hydrolyzing the PPi as energy, thus enabling to transport other important ions and metabolites through the biomembrane. Though the research of H+-PPase in plants has been conducted, H+-PPase of Clostridium tetani (CtH+-PPase) was selected as the model for further studies in this thesis. Previous studies indicated that tyrosine residues play an important role in proton translocation and its hydroxyl group in the functional group is able to accept and release protons. We thus replaced nineteen tyrosine residues in CtH+-PPase individually by alanine with the site-directed mutagenesis technique and analyzed their hydrolysis, proton-translocation and coupling ratio. The enzymatic activities of mutants on Y175, Y226, Y392, Y414 and Y471 were significantly decreased. These five tyrosine residues are highly-conserved in several species. Three dimensional structure suggests they also surround the proton channel of CtH+-PPase. Therefore, we speculated these five positions were involved in the catalytic activity. We then substituted these five tyrosine residues with other amino acids. The enzymatic activities of Y414S and Y414T were restored to approximately 75% of wild-type so that in this position the hydroxyl group is important to CtH+-PPase. From ion effects study, Y414 was also found to be associated with Na+-binding. In conclusion, the functional role of tyrosine residues in CtH+-PPase was substantially elucidated in our study.

References

Baltscheffsky, H., Baltscheffsky, M., Von Stedingl, L. (1969) Inorganic pyrophosphatase, bacterial phosphorylation and evolution of biological energy transformation. In Progress in Photosynthesis Res, Metzner H ed, Internatl. Clarion of Biol. Science, Tubingen. 1313-1318

Baykov, A. A., Hyytia, T., Volk, S. E., Kasho, V. N., Vener, A. V., Goldman, A., Lahti, R., Cooperman, B. S. (1996) Catalysis by E. coli inorganic pyrophosphatase: pH and Mg2+ dependence. Biochemistry 35: 4655-4661

Baykov, A.A., Bakuleva, N.P., Rea, P. A. (1993) Steady-state kinetics of substratehydrolysis by vacuolar H+-pyrophosphatase. A simple three-state model, Eur. J. Biochem. 217:755-762

Carden, D. E., Walker, D. J., Flowers, T. J., and Miller, A. J. (2003) Single-cell measurements of the contributions of cytosolic Na+ and K+ to salt tolerance. Plant physiol. 131:676-683

Drozdowicz, Y. M. and Rea, P. A. (2001) Vacuolar H+-pyrophosphatases: from the evolutionary backwaters into the mainstream. Trends Plant Sci. 6:206-211

Hsiao, Y. Y., Van, R. C., Hung, S. H., Lin, H. H., and Pan, R. L. (2004) Roles of histidine residues in plant vacuolar H+-pyrophosphatase. Biochim. Biophys. Acta 1608:190-199

Hsiao, Y. Y., Pan, Y. J., Hsu, S. H., Huang, Y. T., Liu, T. H., Lee, C. H., Lee, C. H., Liu, P. F., Chang, W. C., Wang, Y. K., Chien, L. F., and Pan, R. L. (2007) Functional roles of arginine residues in mung bean vacuolar H+-pyrophosphatase. Biochim. Biophys. Acta 1767: 965-973

Huang, Y. T., Liu, T.H., Chen, Y. W., Lee, C. H., Chen, H. H., Huang, T. W., Hsu, S. H., Lin, S. M., Pan, Y. J., Lee, C. H., Hsu, I. C., Tseng, F. G., Fu, C. C., and Pan, R. L. (2010) Distance variations between active sites of H+-pyrophosphatase determined by fluorescence resonance energy transfer. J. Biol. Chem. No. 31, pp. 23655-23664

Li, J., Yang, H., Peer, W.A., Richter, G., Blakeslee, J., Bandyopadhyay, A., Titapiwantakun, B., Undurraga, S., Khodakovskaya, M., Richards, E. L., Krizek, B., Murphy, A. S., Gilroy, S., Gaziola, R. A. (2005) Arabidopsis H+-PPase AVP1 regulates auxin-mediated organ development, Science 310:121-125

Lee, C. H., Pan, Y. J., Huang, Y. T., Liu, T. H., Hsu, S. H., Lee, C. H.,
Chen, Y. W., Lin, S. M., Huang L. K., and Pan, R. L. (2011) Identification of essential lysines involved in substrate binding of vacuolar H+-pyrophosphatase. J. Biol. Chem. No. 14, pp. 11970-11976

Maeshima, M. and Yoshida, S. (1989) Purification and properties of vacuolar membrane proton-translocating inorganic pyrophosphatase from mung bean. J. Biol. Chem. 264:20068-20073

Maeshima, M. (2000) Vacuolar H+-pyrophosphatase. Biochim. Biophys. Acta 1465:37-51

Maeshima, M. (2001) Tonoplast transporters: organization and function. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52:469-497

Pan, Y. J., Lee, C. H., Hsu, S. H., Huang, Y. T., Lee, C. H., Liu, T. H., Chen, Y. W., Lin, S. M., Pan, R. L. (2011) The transmembrane domain 6 of vacuolar H+-pyrophosphatase mediates protein targeting and proton transport. Biochim. Biophys. Acta 1807:59-67

Rea, P. A., Pool, R. J. (1993) Vacuolar H+-translocating pyrophosphatases. Annu. Rev. Plant Physiol.44:157-180

Gordon-Weeks, R., Steele, S. H., Leigh, R. A. (1996) The role of magnesium, pyrophosphate, and their complexes as substrates and activators of thevacuolar H+-pumping inorganic pyrophosphatase, (studies using ligand protection from covalent inhibitors), Plant Physiol. 111:195-202

Zhen, R. G., Kim, E. J., Rea, P. A. (1997) Acidic residues necessary forpyrophosphate-energized pumping and inhibition of the vacuolar H+- pyrophosphatase by N,N’-dicyclohexylcarbodiimide, J. Biol. Chem. 272:
22340-22348

Park, S., Li, J., Pittman, J. K., Berkowitz, G. A., Yang, H., Undurraga, S., Morris, J., Hirschi, J. D., Gaziola, R. A. (2005) Up-regulation of a H+-pyrophosphatase (H+-PPase) as a strategy to engineer drought-resistant crop plants, Proc. Natl. Acad. Sci. U.S.A. 102:18830-18835

Yang, S. J., Jiang, S. S., Van ,R. C., Hsiao, Y. Y., and Pan, R. L. (2000) A lysine residue involved in the inhibition of vacuolar H+-pyrophosphatase by fluorescein 5’-isothiocyanate. Biochim. Biophys. Acta 1460:375-383

Yang, S. J., Jiang, S. S., Tzeng, C. M., Kuo, S. Y., Hung, S. H., Pan, R. L. (1996) Involvement of tyrosine residue in the inhibition of plant vacuolar H+-pyrophosphatase by tetranitromethane. Biochim. Biophys. Acta 1294:89-97

Nakanishi, Y., Saijo, T., Wada, Y. and Maeshima, M. (2001) Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase, J. Biol. Chem.276:7654-7660