Abstract
Membrane-integral proton-translocating pyrophosphatase (H+-PPase; EC3.6.1.1) hydrolyzes pyrophosphate (PPi) and uses the energy to create the electrochemical proton gradient cross the membrane. Many studies indicated that tyrosine plays several functional roles in variety of enzymes, such as the phosphorylation site of post-translational regulation, the sorting signal of transmembrane proteins targeting, and direct involvement in enzymatic activity. To determine the critical functional roles of tyrosines in H+-PPase, all tyrosine residues from Clostridium tetani E88 H+-PPase were substituted with alanine by site-directed mutagenesis in Cys-less template and their functional properties examined. We found that 4 variants (Y226A, Y392A, Y414A and Y471A mutants) could not be expressed properly. Another mutant with normal expression, Y175A mutant, displayed deterioration in PPi hydrolysis activity. Moreover, we substituted these five tyrosines to other kinds of amino acids. All variants of Tyr → Phe or Tyr → Trp could be expressed as well as Cys-less mutant. Among these mutants, Y414S and Y414T exhibited a higher PPi hydrolysis activities compared to other substitutions, suggesting that the hydroxyl group is essential for the enzymatic activity. Furthermore, Y414S and Y414T mutants showed the distinct sensitivity of K+-stimulation and Na+-inhibition from Cys-less background. Based on the mutagenesis results and predicted structure, we speculate that Tyr-414 formed a hydrogen bond with Leu-441, which is essential for maintaining the structure of K+-binding site. Taken together, several unexpected functions of tyrosine residues in H+-PPase were revealed in this study.