Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase.

Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization. The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.

Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers.

Catalysis by Escherichia coli inorganic pyrophosphatase (E-PPase) was found to be strongly modulated by Tris and similar aminoalcoholic buffers used in previous studies of this enzyme. By measuring ligand-binding and catalytic properties of E-PPase in zwitterionic buffers, we found that the previous data markedly underestimate Mg(2+)-binding affinity for two of the three sites present in E-PPase (3.5- to 16-fold) and the rate constant for substrate (dimagnesium pyrophosphate) binding to monomagnesium enzyme (20- to 40-fold). By contrast, Mg(2+)-binding and substrate conversion in the enzyme-substrate complex are unaffected by buffer. These data indicate that E-PPase requires in total only three Mg2+ ions per active site for best performance, rather than four, as previously believed. As measured by equilibrium dialysis, Mg2+ binds to 2.5 sites per monomer, supporting the notion that one of the tightly binding sites is located at the trimer-trimer interface. Mg2+ binding to the subunit interface site results in increased hexamer stability with only minor consequences for catalytic activity measured in the zwitterionic buffers, whereas Mg2+ binding to this site accelerates substrate binding up to 16-fold in the presence of Tris. Structural considerations favor the notion that the aminoalcohols bind to the E-PPase active site.

[Characteristics of the immobilization and transformation of sterols by Mycobacterium phlei 1026 cells incorporated into PAAG]. / Izuchenie osobennostei immobilizatsii i transformatsii sterinov vkliuchennymi v PAAG kletkami Mycobacterium phlei 1026.

Experiments were carried out to study the effect of polymerization conditions and polyacrylamide gel structure on the enzyme activity of Mycobacterium phlei immobilized cells degrading sterols to C19-steroids. The modified sterol 4-chlostene-3(O-carboxymethyl)oxime was used as substrate. The polymerization conditions (the number of the cells incorporated and the reaction temperature) significantly affected the ability of the immobilized cells to degrade the sterol side chain. The monomer contact produced no negative effect on the cellular enzymic activity. The following conditions of immobilization and transformation appear to be optimal: cell incorporation into 10% gel, monomer (AA:MBA) ratio 95:5, polymerization temperature 2-8 degrees C, polymerization mixture biomass 40 mg/ml, granule size about 1 mm. Enzymic activity can be substantially increased via cell incubation in the nutrient medium.