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

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Abstract
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, MgP1, protect the enzyme against inactivation. Ions of metal‐activators (Mg2+, Zn2+) exert no influence on the inactivation rate. On the contrary, the Ca2+‐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 pK 2 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 Cn2+ pyrophosphatase complex.