Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase

Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PPi hydrolysis (pH 4.8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4.8-9.3), equilibrium formation of enzyme-bound PPi (pH 4.8-9.3), and Mg2+ binding (pH 5.5-9.3). These data confirmed that enzyme-PPi intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PPi release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PPi intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PPi intermediate decelerates PPi hydrolysis but accelerates PPi release by affecting the back isomerization. The binding of two Mg2+ ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) similar to 9 is modulatory in PPi hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.