Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase

Five catalytic functions of yeast inorganic pyrophosphatase were measured o ver 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 formati on of enzyme-bound PPi (pH 4.8-9.3), and Mg2+ binding (pH 5.5-9.3). These d ata confirmed that enzyme-PPi intermediate undergoes isomerization in the r eaction cycle and allowed estimation of the microscopic rate constant for c hemical bond breakage and the macroscopic rate constant for PPi release. Th e isomerization was found to decrease the pK(a) of the essential group in t he enzyme-PPi intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PPi intermediate decelerates PPi hydr olysis but accelerates PPi release by affecting the back isomerization. The binding of two Mg2+ ions to free enzyme requires about five basic groups w ith a mean pK(a) of 6.3. An acidic group with a pK(a) similar to 9 is modul atory in PPi hydrolysis and metal ion binding, suggesting that this group m aintains overall enzyme structure rather than being directly involved in ca talysis.