Tyrosine-89 is important for enzymatic activity of S. cerevisiae inorganic pyrophosphatase.

7-Chloro-4-nitro-benzofurazan selectively modifies one PPase Tyr residue per subunit and lowers the enzyme activity. Hydrolysis of the modified protein by trypsin and then by chymotrypsin produces the 82-89 peptide which possesses modified Tyr-89. Substrate analog (CaPPi) and the product of the enzyme reaction, MgPi, protect the enzyme against inactivation. Ions of metal-activators (Mg2+, Zn2+) exert no influence on the inactivation rate. On the contrary, the Ca(2+)-inhibitor of the enzyme accelerates the reaction by binding to the high-affinity site, and effectively decreases it when Ca2+ binds to both sites. Mg2+ competes with Ca2+ for one binding site, which is the low affinity site for Mg2+ and the high-affinity site for Ca2+. The Ca2+ saturation of the high-affinity site decreases the pK2 of Tyr-89, probably due to direct coordination between Tyr and Ca2+. The observed properties of Tyr-89 modification enable us to propose that Tyr-89 serves as a proton donor for phosphate releasing during enzymatic hydrolysis of pyrophosphate. The Ca2+ inhibitory effect on the enzyme activity may be due to the existence of a Tyr-89 bond in the Ca2+ pyrophosphatase complex.