Mutagenic Analysis of Histidine-716 of Vacuolar H+-pyrophosphatase

• Main Author: 李保蓁
• Other Authors: Rong-Long Pan
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/51996682456066924689

碩士 === 國立清華大學 === 生命科學系 === 90 === 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.