PubMed Health⌕ Search

Biomedical subjects

S M Avaeva

Publications and source records attributed to S M Avaeva.

11 recordsLinked to original sources
PubMed1979

Fluoride inhibition of inorganic pyrophosphatase. IV. Evidence for metal participation in the active center and a four-site model of metal effect on catalysis.

Atomic spectroscopy of native yeast inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) after gel filtration showed that it only binds activating Mg2% in an easily dissociable manner. Formation of a covalent intermediate between the enzyme and an entire substrate molecular in the presence of fluoride, however, dramatically strengthened the binding of two Mg2+ per subunit and eliminated at neutral pH the effect of added metals on protein fluorescence but not on the absorption spectrum, suggesting that different mental binding sites influence the two spectra. This conclusion was confirmed by spectra studied on native enzyme. A third, low-affinity site for Mg2+ was found on the enzyme pH greater than 8. A model of enzyme-substrate-metal interactions was proposed, according to which the fluorescence-controlling site belongs to the active center and substrate can only be bound to it as a 1 : 1 complex with metals.

Binding Sites↗
PubMed1978

[Catalytic properties of three isoenzymes possessing pyrophosphatase activity, isolated from baker's yeast].

A kinetic study of inorganic pyrophosphatase isolated from brewer's yeast was done. It was shown that all three isoenzymes have the same pH-optimum and specificity with respect to substrate and metal activator. Statistical treatment of the kinetic data yielded equilibrium and catalytical constants, describing enzyme interaction with the metal activator and substrate. The catalytic properties of all three isoenzymes are similar to those of the baker's yeast pyrophosphatase. The fluoride inhibition pattern for inorganic pyrophosphatase from brewer's yeast is similar to that for the baker's yeast enzyme.

Catalysis↗
PubMed1977

The essential activated carboxyl group of inorganic pyrophosphatase.

1. A carboxyl group of high reactivity has been found in inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from yeast. This group interacts with agents which react neither with carboxyl groups of low molecular weight compounds nor with other carboxyl groups of the protein. 2. The reaction of this activated carboxyl group with inorganic phosphate, hydroxylamine, N-methyl- and O-methylhydroxylamines, and glycine methyl ester has been studied. 3. Homoserine and homoserine lactone were found in the hydrolyzate of phosphorylated and NaBH4-reduced pyrophosphatase, indicating that an aspartyl residue is phosphorylated. 4. Hydroxylamine and other nucleophilic agents cause inactivation of pyrophosphatase as a result of interaction with a carboxyl group. Both diaminobutyric and diaminopropionic acids were seen in the acid hydrolyzate of the protein treated with hydroxylamine and subjected to rearrangement in the presence of carbodiimide. 5. The ways in which the activation of a carboxyl group in the enzyme is achieved and the presumed mechanism of action of inorganic pyrophosphatase are discussed.

Carbodiimides↗
PubMed1977

Fluoride inhibition of inorganic pyrophosphatase. II. Isolation and characterization of a covalent intermediate between enzyme and entire substrate molecule.

A presumed pyrophosphoryl-enzyme intermediate of the reaction catalyzed by bakers' yeast inorganic pyrophosphatase pyrophosphate phosphohydrolase, EC 3.6.1.1) has been isolated using fluoride-mediated inactivation of the enzyme during catalysis. The analysis of the F--inactivated pyrophosphatase revealed the presence of one molecule of PPi and one atom of fluoride per active site. The incubation of the inactivated enzyme at 25 degrees C and pH 7.2 resulted in gradual recovery of catalytic activity and concomitant removal of PPi by a first-order reaction with tau1/2 of 1 h. The digestion of the F--treated pyrophosphatase with pepsin yielded phosphorous-containing peptides, which were reduced with NaBH4 and gave homoserine and homoserine lactone after acid hydrolysis. This suggests that the PPi residue is linked to the protein through a bond of an acyl phosphate type involving the beta-COOH function of aspartic acid. Together with the results of the kinetic studies of fluoride inhibition of pyrophosphatase reported in accompanying papers, these findings strongly indicate that the enzyme-substrate compound stabilized by fluoride is a transient of the catalytic reaction.

Binding Sites↗
PubMed1977

Fluoride inhibition of inorganic pyrophosphatase. III. Dependence on the nature of substrate and metal ion cofactor.

Studies of fluoride interaction with bakers' yeast inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) in the presence or absence of enzyme-catalyzed reactions have revealed pronounced specificity of this inhibitor. It was found that the inhibition of enzymic hydrolysis of PPi, ADP, ATP and tripolyphosphate in the presence of Zn2+ and Mn2+ at pH 6.5 is not time dependent and by far less extensive as that observed for the Mg2+-stimulated cleavage of PPi (apparent Ki values differ by three orders of magnitude). Addition of Ca2+ to the latter reaction decrease proportionally the activity of the enzyme and the rate constant for the binding of fluoride to it, which indicates that the enzyme-substrate complexes containing both Mg2+ and Ca2+ are inert in the reaction with fluoride. Preincubation of pyrophosphatase with NaF and various metal cations and substrates, in conditions where the enzyme does not act as a catalyst, does not affect its activity compared to controls lacking fluoride. The results are consistent with the proposed mechanism of mutual hindrance of substrate and fluoride release from the active site of pyrophosphatase.

Binding Sites↗
PubMed1977

[Comparative kinetic studies of Mg2+-activated hydrolysis of tripolyphosphate and pyrophosphate by inorganic pyrophosphatase].

Computer analysis of P3 and pyrophosphate conversion rate dependence on substrate and metal-activator concentrations reveals the identity of kinetic patterns. Dissociation and catalytical constants for the enzyme combinations with two types of metal-substrates complexes, MS and M2S, at pH 9.0 are by one to two orders of magnitude "poorer" for P3 as compared to PPi. Optimal pH value for the hydrolysis of P3 is by 2 units higher than this for the hydrolysis of PPi. pH profiles for the kinetic parameters in the pH range 8.0--9.5 differ considerably for the two substrates, presumably due to the existence of additional catalitically important ionisations in the reaction with P3.

Diphosphates↗
PubMed1976

Fluoride inhibition of inorganic pyrophosphatase. I. Kinetic studies in a Mg2+-PPi system using a new continuous enzyme assay.

Reversible inhibition of bakers' yeast inorganic pyrophosphatase (EC 3.6.1.1) by fluoride has been studied as a function of substrate, metal-ion activator and inhibitor concentrations and pH using a new continuous enzyme assay with an automatic phosphate analyzer. The inhibition was shown to be the result of tight binding of fluoride by two catalytically active enzyme-substrate complexes. The reaction between pyrophosphatase and fluoride is relatively slow, so that the rate constants for the binding and release of the inhibitor were derived from phosphate formation curves measured on the time scale of enzyme assays. The pH-dependence of the inhibition reaction in the alkaline medium indicates that both the fluoride-enzyme interaction and the catalytic step of the pyrophosphatase reaction are controlled by the same group on the protein. In the acidic medium, the inhibition is considerably enhanced, presumably because of the protonation of another enzyme group.

Autoanalysis↗
PubMedDate not supplied

[Inactivation of yeast inorganic pyrophosphatase by sodium dodecyl sulfate and cetyltrimethyl ammonium bromide].

The kinetics of the inactivation of yeast inorganic pyrophosphatase by sodium dodecyl sulfate (SDS) and cetyltrimethyl ammonium bromide were studied. Micellar forms of detergents were shown to be an active reagetns under conditions tudied. The possible scheme of the inactivation including reversible formation of the micellar-protein complex and subsequent penetration of the bound detergent molecules in the protein is proposed. The enzyme ionogenic group with pK 7-8 by 25 degrees C is found to be responsible for conformational changes of the enzymes. The influence of the specific ligands on inactivation of yeast pyrophosphatase by SDS is studied, and dissociations constants of corresponding enzyme-ligand complexes are calculated.

Cetrimonium Compounds↗