臺灣博碩士論文加值系統

我們已知植物液胞膜上焦磷酸水解酵素(簡稱V-PPase)，在維持細胞質的中性酸鹼度扮演很重要的角色：V-PPase藉由水解焦磷酸驅動質子由細胞質打入液胞中，以維持細胞質的中性酸鹼度。在以diethylpyrocarbonate修飾綠豆V-PPase的研究中，我們已經知道組胺酸可能有參與催化水解的過程(Hsiao, Y. Y., Van, R. C., Hung, H. H., and Pan, R. L. (2002) Journal of Protein Chemistry 21: 51-58)。在對6個V-PPase組胺酸的突變分析中顯示，組胺酸716可能是參與催化過程的胺基酸，在此篇論文，我們利用點突變的方法，將組胺酸716以其他12個胺基酸取代之，並測其活性。結果發現，所有的突變株，不論是焦磷酸的水解活性，或是質子的運送能力均下降﹔H716W與H716F突變株不但仍保有被鉀離子激發的特性，而且還保有60%以上的焦磷酸水解活性及28%以上的質子傳送能力，較保留突變株(H716K與H716R)或相反特性突變株所保有的活性，均高出許多，由此推測，芳香環在這個位置是重要的；突變株及正常的焦磷酸水解酵素的最適水解活性均在pH 7.5，且在熱穩定的實驗中，均在53℃時活性急速下降，表示在716突變不會造成水解區域的結構改變﹔在外加imidazole的實驗中，所有突變株活性的結果非常分歧，推翻組胺酸716是直接參與催化過程的假設。綜合所有實驗的結果，我們推測組胺酸716在V-PPase的C端，會間接的影響焦磷酸水解、質子傳輸、及被鉀離子激發的活性。

Plant vacuolar H+-translocation inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) has been considered to play a significant role on the maintenance of pH value of cytoplasm via the proton translocation from cytosol to vacuolar lumen as being triggered at the expense of PPi hydrolysis. The diethylpyrocarbonate (DEPC)-modification study of Vigna radiata V-PPase has demonstrated the involvement of histidyl residues in or near the catalytic site of the enzyme (Hsiao, Y. Y., Van, R. C., Hung, H. H. and Pan, R. L. (2002) Journal of Protein Chemistry 21: 51-58). The mutagenic analysis of six histidines revealed that His-716 might be the candidate participating in catalytic process. His-716 was substituted with 12 different amino acids and examined the enzymatic properties. All mutants exhibited the diminution of PPi hydrolytic and H+ translocation activities. Only did H716W and H716F V-PPase hold the potassium-stimulated properties and retain more than 60 % of PPi hydrolytic and 28 % of proton transport activity. The conserved (H716K and H716R) or converse (H716D) mutations all lost their enzymatic activities, suggesting the importance of aromatic ring in this position. The optimum PPi hydrolytic activity of V-PPase of wild-type and mutants were all at pH 7.5. Moreover, the PPi hydrolytic activity of all proteins dramatically declined at around 53℃. Therefore, the mutation on His-716 did not affect the conformation of hydrolytic pocket. Imidazole preincubation was used to clarify the role of His-716 in PPi hydrolysis. The distinct outcomes of all mutants after the addition of exogenous imidazole refute the notion that His-716 directly participated in the enzymatic domain. Instead, His-716 plays roles in the long distant or indirect effects on PPi hydrolytic, H+ transport, and K+-sensitive activities on the C-terminus of V-PPase.

Introduction---------------------------------------------1
Materials and Methods------------------------------------5
Results-------------------------------------------------14
Discussion--------------------------------------------- 18
References----------------------------------------------25
Tables and Figures--------------------------------------30

Ädelroth, P., Paddock, M. L., Tehrani, A., Beatty, T., Feher, G., and Okamura, M. Y. (2001) Identification of the proton pathway in bacterial reaction centers: decrease of proton transfer rate by mutation of surface histidine at H126 and H128 and chemical rescue by imidazole identifies the initial proton donors. Biochemistry 40: 14538-14546.
Ausubel, F. M., Brent, R., Kingston, R., Moore, D. D., Seidman, J. G., Smith, J. a., and Struhl, K. (1992) in Short Protocols in Molecular Biology, Unit: 13.7 pp. 31-35, 4th Ed, John Wiley & Sons Inc., Canada.
Barik, S. (1997) in PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering (White, B. A. eds) pp. 173-182, Humana Press Inc., New Jersey, USA.
Bradford, M. (1976) A rapid and sensitive method for the quantities of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72: 248-254.
Chen, C. H. (2001) Functional determination in C-terminal region of vacuolar H+-pyrophosphatase from etiolated mung bean seedlings. Department of Life Science, College of Life Science, National Tsing Hua University,Hsin Chu 30043, Taiwan, ROC.
Chen, J. H. (2000) Roles of C-terminal region of vacuolar H+-pyrophosphatase was determined by deleted mutation. Department of Life Science, College of Life Science, National Tsing Hua University, Hsin Chu 30043, Taiwan, ROC.
Drozdowicz, Y. M., Kissinger, J. C., and Rea, P. A. (2000) AVP2, a sequence-divergent, K+-insenstive H+-translocating inorganic pyrophosphatase from Arabidopsis. Plant Physiol. 123: 353-362.
Drozdowicz, Y. M., and Rea, P. A. (2001) Vacuolar H+ pyrophosphatases: from the evolutionary backwaters into the mainstream. Trends Plant Sci. 6: 206-211.
von Heijne, G. (1992) Membrane protein structure prediction hydrophobicity analysis and the positive-inside rule. J. Mol. Biol. 225: 487-494.
Hsiao, Y. Y., Van, R. C., Hung, H. H., and Pan, R. L. (2002) Diethylpyrocarbonate inhibition of vacuolar H+-pyrophosphatase possibly involves a histidine residue. J. Protein Chem. 21: 51-58.
Huang, S., and Tu., S. C. (1997) Identification and characterization of a catalytic base in bacterial luciferase by chemical rescue of a dark mutant. Biochemistry 36: 14609-14615.
Kim, Y., Kim, E. J., and Rea, P. A. (1994) Isolation and characterization of cDNAs encoding the vacuolar H+-pyrophosphatase of Beta vulgaris. Plant Physiol. 106: 375-382.
Kim E. J., Zhen, R. G., and Rea, P. A. (1994) Heterologous expression of plant vacuolar pyrophospatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport. Proc. Natl. Acad. Sci. USA 91: 6128-6132.
Kim E. J., Zhen, R. G., and Rea, P. A. (1995) Site-directed mutagenesis of vacuolar H+-pyrophosphatase. Necessity of Cys634 for inhibition by maleimides but not catalysis. J. Biol. Chem. 270: 2630-2635.
Kirby, A. H., and Neuberger, A. (1938) Glyoxalines: the determination of their pK values and the use of their salts as buffer. Biochem. J. 32: 1146-1151.
Kuo, S. Y., and Pan, R. L. (1990) An essential arginyl residue in the tonoplast pyrophosphatase from etiolated mung bean seedlings. Plant Physiol. 93: 1128-1133.
Lehoux, I. E., and Mitra, B. (1999) (S)-Mandelate dehygenase from Pseudomonas putida: mutations of the catalytic base histidine-274 and chemical rescue of activity. Biochemistry 38: 9948-9955.
Lin, Y. T. (2000) Essential roles of C-terminal domain in vacuolas H+-pyrophosphatase from etiolated mung bean seedlings. Department of Life Science, College of Life Science, National Tsing Hua University, Hsin Chu 30043, Taiwan, ROC.
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.
Maruyama, C., Tanaka, Y., Takeyasu, K. Yoshida, M., and Sato, M. H. (1998) Structural studies of the vacuolar H+-pyrophosphatase: sequence analysis identification of the residues modified by fluorescent cyclohexylcarbodiimide and maleimide. Plant Cell Physiol. 39: 1045-1053.
McPherson, M. J., and Møller, S. G. (2000) in PCR, pp.143-182, BIOS Scientific Publishers Ltd., Oxford, UK.
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(10): 7654-7660.
Newmyer, S. L., Sun, J., Loehr, T. M., and de Montellano, P. R. O. (1996) Rescue of the horseradish peroxidase His-170  Ala mutant activity by imidazole: importance of proximal ligand tethering. Biochemistry 35: 12788-12795.
Palmgren, M. G. (1991) Acridine orange as a probe for measuring pH Gradients across membranes: mechanism and limitations. Anal. Biochem. 192: 316-321.
Salisbury, F. B., and Ross, C. W. (1992) Plant Physiology, pp. 24-25.4th edition, Wadsworth Inc., California, USA.
Tu, C. K., and Silverman, D. N. (1989) Role of histidine 64 in the catalytic mechanism of human carbonic anhydrase II studies with a site-specific mutant. Biochemistry 28: 7913-7918.
van Veldhoven, P. P., and Mannaerts, G. (1987) Inorganic and organic phosphate measurements in the nanomolar range. Anal. Biochem. 161:45-48.
De Wall, S. L., Meadows, E. S., Barbour, L. J., and Gokel, G. W. (1999) Synthetic receptors as models for alkali metal cation- binding in proteins. Proc. Natl. Acad. Sci. USA 97: 6271-6276.
Weibe C. A., Dibattista, E. R., and Fliegel, L. (2001) Functional role of polar amino acid residues in Na+/H+ exchangers. Biochem. J. 357: 1-10.
Yang, S. J., Jiang, S. S., Tzeng, C. M., Kuo, S. Y., Hung, S. H., and Pan, R. L. (1996) Involvement of tyrosine residues in the inhibition of plant vacuolar H+-pyrophosphatase tetranitromethane. Biochim. Biophys. Acta 1294:89-97.
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.
Zhen, R. G., Kim, E. J., and Rea, P. A. (1997) Acidic residues necessary for pyrophosphatase-energized pumping and inhibition of the vacuolar H+-pyrophosphatase by N, N’-dicyclohexylcarboiimide. J. Biol. Chem. 272: 22340-22348.