| Summary: | 博士 === 國立清華大學 === 生命科學系 === 93 === Abstract
Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase containing a single type of polypeptide with a molecular mass of approximately 81 kDa. Topological analysis tentatively predicts that mung bean V-PPase contains 14 transmembrane domains. Alignment analysis of V-PPase demonstrates that the transmembrane domain 5 (TM5) of the V-PPase is highly conserved and located at the N-terminal side of the putative substrate-binding loop. Furthermore, the hydropathic analysis of V-PPase showed a relatively lower degree of hydrophobicity in TM5 region than others. Accordingly, it is believed that the TM5 is probably involved in the proton translocation of V-PPase. In this study, we used the site-directed mutagenesis to examine the functional role of amino acid residues in TM5 of V-PPase. A series of mutants singly replaced by alanine residues along TM5 was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Our results indicate that the several mutants, attributed as Group 1, displayed minor variations in enzymatic properties, while the others, attributed as Group 2 including those mutated at E225, a GYG motif (residues from 229 to 231), A238, and R242, showed a serious decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase. The mutant Y230A also displayed a relief in the cation effects on the V-PPase. Mutation of several residues along TM5 brought as well about the structural changes of V-PPase. Analysis on sequence alignment suggests that GYG motif may form a cation-binding cage for V-PPase. A model with two helical wheels on both sides of GYG motif is thus proposed for TM5 of V-PPase. Two more mutations of Y230 (Y230S and Y230F) were constructed to investigate the structure/function role of this tyrosine residue in detail. The substitution of tyrosine to phenylalanine at 230 did not change the hydrolytic activity of V-PPase much, but shifted the proton translocating ability, hence the coupling efficiency of V-PPase to 60% of the wild type. The hydroxyl moiety on the Y230 residue might be therefore directly involved in the proton translocating process of V-PPase.
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