Tryptic hydrolysis of inorcanic pyrophosphate modified by phosphate and O-phosphoethanolamine

By the peptide map method, a phosphorylated peptide has been isolated from a tryptic hydrolysate of phosphorylated yeast inorganic pyrophosphatase (I), and this is a direct proof of the formation of a covalent bond between (I) and phosphate in the course of this reaction. The isolation and analysis of the peptide from the tryptic hydrolysate shows that the phosphate acceptor is probably the aspartic acid residue 240 or 248. Analysis of a tryptic hydrolysate of (I) modified with O-phosphoethanolamine has shown that O-phosphoethanolamine forms an amide bond with the carboxy group of the same aspartic acid residue. In an alkaline medium, the phosphate residue migrates to the imidazole ring of a histidine residue, apparently that present in position 222.     

Reaction of yeast inorganic pyrophosphatase with inorganic phosphate labeled with radioactive phosphorus

Conclusions The reaction of yeast inorganic pyrophosphatase with inorganic [p³²]phosphate forms a phosphorylated enzyme.     

Hydrolysis of the pyrophosphate bond of seryl pyrophosphates by the alkaline phosphatase ofEscherichia coli

Summary It has been shown that the pyrophosphate bond of seryl pyrophosphates is cleaved by the alkaline phosphatase ofE. coli.Khimiya Prirodnykh Soedinenii, Vol. 3, No. 5, pp. 328–331, 1967     

The phosphorylation of carboxylic acids and amino acids by diseryl pyrophosphates

Summary 1. From the reaction of chymotrypsin with the NPE of CBZ-L- and CBZ-D-valine the enzyme-substrate compounds CBZ-L-valylchymotrypsin and CBZ-D-valylchymotrypsin have been isolated. The methyl esters of benzoyl-D-phenylalanine and of benzoyl-L-valine also form enzyme-substrate compounds with chymotrypsin, although to a smaller extent. The methyl ester of benzoyl-D-valine does not react with chymotrypsin.2. Conditions for the isolation of a water-soluble enzyme-substrate compound have been found.Khimiya Prirodnykh Soedinenii, Vol. 2, No. 2, pp. 134–138, 1966Given at the First All-Union Biochemical Congress, 25 January 1964 (Leningrad).     

Some features of hydrolysis of organic and inorganic substrates by Escherichia coli inorganic pyrophosphatase in the presence of various activator cations

The kinetics of hydrolysis of the inorganic (PP_i) and organic (ATP) substrates by Escherichia coli inorganic pyrophosphatase (PPase) and its mutant forms with Asp42 replaced by Ala, Asn, or Glu was studied. The Mn²⁺ or Zn²⁺ ions were used as activators of the enzymatic reaction. The kinetic parameters of hydrolysis were determined. The inhibitory effect of these cations on substrate hydrolysis was investigated. The dissociation constants were calculated for the Mn²⁺- and Zn²⁺-binding activator and inhibitor subsites of E. coli PPase. The observed hydrolysis rate of PP_i increases in the series Zn²⁺ < Mn²⁺ < Mg²⁺, whereas the potential efficiency of these cations decreases in this series. Hydrolysis of ATP by E. coli PPase occurs only in the presence of Mn²⁺. The reasons for the observed differences in the substrate specificity of the enzyme are discussed.