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

[摘要]

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 (CaPP_i) 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.

[学科分类] 生物化学/生物物理

[关键词] Inorganic pyrophosphatase;Chemical modification;Essential groups;Me2+ affinity;S. cerevisiae