| Summary: | 碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 100 === 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.
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