[Inhibition of inorganic pyrophosphatase from Escherichia coli with inorganic phosphate]. / Ingibirovanie neorganicheskoi pirofosfatazy Escherichia coli neorganicheskim fosfatom.

The interaction of inorganic pyrophosphatase from E. coli with inorganic phosphate (Pi) was studied in a wide concentration range of phosphate. The apoenzyme gives two inactive compounds with Pi, a product of phosphorylation of the carboxylic group of the active site and a stable complex, which can be detected in the presence of the substrate. The phosphorylation occurs when Pi is added on a millimole concentration scale, and micromole concentrations are sufficient for the formation of the complex. The formation of the phosphorylated enzyme was confirmed by its sensitivity to hydroxylamine and a change in the properties of the inactive enzyme upon its incubation in alkaline medium. The phosphorylation of pyrophosphatase and the formation of the inactive complex occur upon interaction of inorganic phosphate with different subsites of the enzyme active sites, which are connected by cooperative interactions.

[Irreversible specific inhibition of E. coli inorganic pyrophosphatase with amines]. / Neobratimoe spetsificheskoe ingibirovanie neorganicheskoi pirofosfatazy E. coli aminami.

An unusually high reactivity of the carboxyl groups of the active site of E. coli inorganic pyrophosphatase towards amines was shown. Amino acid esters and other amines are specific irreversible inhibitors of the enzyme. The reaction involves the formation of an enzyme-inhibitor complex followed by the chemical modification of dicarboxylic amino acid residues. It is assumed that the binding of the positively charged inhibitor occurs at the binding site of cations-activators.

[Functionally important residues of glutamic acid in E. coli pyrophosphatase. I. Chemical modification and localization in the primary structure]. / Funktsional'no vazhnyi ostatok glutaminovoi kisloty v pirofosfataze E. coli. I. Khimicheskaia modifikatsiia i lokalizatsiia v pervichnoi strukture.

Inorganic pyrophosphatase of E. coli is rapidly and irreversibly inactivated by 5-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K). The appearance in the absorption spectrum of a maximum at 340 nm testifies to the formation of an enzyme enol ester with the inhibitor. The non-hydrolyzable substrate analog CaPP1 partly protects the enzyme from inactivation. A peptide has been isolated from a tryptic hydrolysate of inactivated enzyme which contains an amino acid residue whose modification is critical for the enzyme activity. This peptide corresponds to residues 95-104 of pyrophosphatase and contains four dicarboxylic acid residues. A peptide containing a modified glutamic acid residue was isolated from modified pyrophosphatase hydrolyzed by protease v8. This peptide represents a fragment of a tryptic modified peptide and has a Glu-Ala-Gly-Glu (residues 98-1C1) structure. It is concluded that inactivation of E. coli pyrophosphatase by Woodward's reagent K is a result of selective modification of Glu98, apparently by the most reactive dicarboxylic amino acid within the enzyme active center.

[Functionally important tyrosine residues in Saccharomyces cerevisiae pyrophosphatase. I. Chemical modification and localization in the primary structure]. / Funktsional'no vazhnyi ostatok tirozina v pirofosfataze S. cerevisiae. I. Khimicheskaia modifikatsiia i lokalizatsiia v pervichnoi strukure.

Inorganic pyrophosphatase (PPase) of S. cerevisiae is effectively inactivated by 7-chloro-4-nitrobenzofuran; the CaPP1 substrate analog has a protective effect. The modified enzyme separated from low molecular weight contaminants has an adsorption maximum at 345 nm. Preliminary modification of PPase SH-groups does not influence the enzyme binding to the inhibitor. The PPase activity is reconstituted by beta-mercapto-ethanol; hence, the inhibiting effect of the reagent is due to modification of tyrosine residues. A single reagent-containing peptide was isolated by specific adsorption from the tryptic hydrolysate of modified PPase. Within the primary structure of PPase, this peptide occupies positions 82-111 and contains two tyrosine residues. Hydrolysis of the isolated peptide by chymotrypsin and determination of the structure of fragments obtained by mass spectrometry and automated sequencing revealed that inactivation of PPase is due to selective modification of Tyr89.

[A functionally important Tyr-89 residue in Saccharomyces cerevisiae pyrophosphatase. II. A possible role in the mechanism of enzyme action]. / Funktsional'no vazhnyi ostatok Tyr-89 pirofosfatazy S. cerevisiae. II. Vozmozhnaia rol' v mekhanizme deistviia fermenta.

Earlier it has been demonstrated that inactivation of inorganic pyrophosphatase (PPase) of S. cerevisiae by 7-chloro-4-nitronbenzofurasane is due to modification of Tyr89. The effect of pH and active center ligands on this reaction has been studied. It was found that pK for Tyr89 does not exceed 8.5; the phosphate-metal complex binding to the high affinity center protects Tyr89 from inactivation. Activating ions (Mg2+ and Zn2+) do not influence the inactivation, whereas the PPase inhibitor, Ca2+, enhances this process after saturation of the high affinity binding site. Saturation of two binding sites with Ca2+ has a protective effect on the enzyme. An increase in the rate of Tyr89 binding to the inhibitor in the presence of low concentrations of Ca2+ is due to the decrease of Tyr89 pK. The data obtained suggest that Tyr89 is located near the high affinity binding site for phosphate. The high reactivity of Tyr89 and its tight binding in the active center point to the presence of a hydrogen bondage with the substrate and suggest a role of a proton donor whose acceptor is the product of the enzymatic reaction, i.e., phosphate.

[Functionally important lysine residues in inorganic pyrophosphatase from E. coli. I. Interaction of inorganic pyrophosphatase with pyridoxal-5'-phosphate]. / Funktsional'no vazhnyi ostatok lizina v neorganicheskoi pirofosfataze iz E. coli. I. Vzaimodeistvie neorganicheskoi pirofosfatazy s piridoksal'-5'-fosfatom.

Interaction of inorganic pyrophosphatase from E. coli with pyridoxal-5'-phosphate includes binding of the reagent at the active site through the phosphate group and then a reversible modification of one lysine residue in each of the enzyme's subunit. In the equilibrium state the protein's molecules contain both inactive modified and native subunits. A stable secondary amine is formed upon the sodium borohydride reduction of the modified protein.

[Functionally important lysine residues in inorganic pyrophosphatase from E. coli. II. Isolation and characteristics of modified tryptic peptide and analysis of the functional role of lysine residues controlling enzyme activity]. / Funktsional'no vazhnyi ostatok lizina v neorganicheskoi pirofosfataze iz E. coli. II. Vydelenie i kharakteristika modifitsirovannogo tripticheskogo peptida i issledovanie funktsional'noi roli ostatka lizina, kontroliruiushchego fermentativnuiu aktivnost'.

Inactivation of inorganic pyrophosphatase from E. coli by pyridoxal-5'-phosphate is due to the modification of a lysine residue located in the tryptic peptide with the Asp-Leu-Pro-Glu N-terminal sequence. In course of the enzymatic process this lysine-residue appears to be in the protonated state and either operators as the proton donor for the product of the enzymatic reaction or is involved in stabilization of the transition state.

[Monomeric form of E. coli pyrophosphatase. Regulatory properties, stability and interaction with affinity inhibitors]. / Monomernaia forma pirofosfatazy E. coli. Reguliatornye svoistva, stabil'nost' i vzaimodeistvie s affinnymi ingibitorami.

A comparative study on the E. coli inorganic pyrophosphatase subunit and native oligomer disclosed that the quaternary structure is not an essential prerequisite for exhibiting by the enzyme its regulatory properties. However, association of the subunits enhances the stability of the protein molecule and is obligatory for irreversible affinity inhibition.

[Interaction of inorganic pyrophosphatase from yeast with alkylated derivatives of Sepharose]. / Vzaimodeistvie neorganicheskoi pirofosfatazy iz drozhzhet s alkilirovannymi proizvodnymi sefarozy.

The adsorption of inorganic pyrophosphatase from baker's yeast by alkylated polysaccharides involves both electrostatic and hydrophobic interactions. The enzyme is eluted by NaCl solutions of different ionic strengths depending on the adsorbent hydrophobicity. The degree of purification on different adsorbents varies from 2- to 80-fold.

[Affinity labeling of inorganic pyrophosphatase from yeast by methylphosphate]. / Affinnaia modifikatsiia neorganicheskoi pirofosfatazy iz dorzhzhei metilfosfatom.

Yeast inorganic pyrophosphatase is specifically and irreversibly inactivated by methylphosphate. The high rate of inhibition, the protective effect of the substrate, the strict correlation between the degree of inhibition and the amount of the protein-bound reagent and the effect of saturation of the enzyme with methylphosphate provide evidence in favour of the reaction in the active center. Modification of two chemically identical enzyme subunits proceeds at different rates and results in a formation of phosphorylated subunits with different stability of the phosphate bond, which is indicative of the mutual effects of the pyrophosphatase subunits. The reaction between the modified enzyme and hydroxylamine suggests that the interaction between pyrophosphatase and methylphosphate entails modification of the carboxylic groups of two active centers, resulting in a formation of the acylphosphate bonds.

[Preparation of peptide maps of sheep fetus haptoglobin using immobilized trypsin]. / Poluchenie peptidnykh kart gaptoglobina plodov ovets s ispol'zovaniem immobilizovannogo tripsina.

A comparison of peptide maps of sheep foetus haptoglobin (Hp) phenotypes revealed a significant difference in 6 peptides. The map of multicomponent Hp2 shows the presence of a group of neutral peptides, which was absent in the case of a single-component Hp1. This difference is probably due to the presence of the alpha-subunit in Hp2.

[Reconstruction of native bakers' yeast inorganic pyrophosphatase from its subunits]. / Rekonstruktsiia kataliticheski aktivnogo fermenta iz sub'edinits drozhzhevoi neorganicheskoi pirofosfatazay.

The subunits of inorganic pyrophosphatase were obtained both in the presence of 4 or 6 M Gu--HCl and in an alkaline medium at pH 10.5. The reassociation of the subunits into an active dimeric form was carried out using dialysis. After reconstruction the enzyme exhibits the same activity as does native pyrophosphatase.