Purification and characterization of the multiple forms of β-galactosidase of Escherichia coli

Marchesi, Sally L.; Steers, Edward; Shifrin, Sidney

doi:10.1016/0005-2795(69)90223-2

Cited by 59 publications

(16 citation statements)

References 15 publications

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“…Stability decreased sharply below pH 6 and more gradually above pH 8 (Wallenfels and Malhotra 1961). At pH 3.5 and 11.5, the enzyme was completely dissociated into inactive monomers (Marchesi et al 1969).…”

Section: Effect Of Ph On Stabilitymentioning

confidence: 99%

Stability and Enzymatic Properties of ?-Galactosidase From Kluyveromyces Fragilis

Mahoney

Whitaker

1978

J Food Biochemistry

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Kluyveromyces fragilis β‐galactosidase purified to electrophoretic, chromatographic and immunochemical homogeneity was used. The enzyme specifically required potassium ions for stability; MnCl2 increased the stability. The enzyme was maximally stable at pH 6.5 to 7.5; stability was markedly less at pH's below 6.5 and above pH 8.5 at 37°C. Temperature denaturation followed first order kinetics with an activation energy for denaturation of 56 kcal/mol. Maximum activity was achieved in the presence of 5mM KCl. In potassium phosphate buffer, the enzyme was further activated by Mn2+, Mg2+, Co2+ and Zn2+; Mn2+, at 0.1 mM, gave the highest activation. None of these ions activated the enzyme in Tris buffer and> 0.1 mM Zn2+ caused complete loss of activity. Activity was completely inhibited by ethylenediaminetetraacetate and partially restored by addition of MnCl2. p‐Chloromercuribenzoate caused rapid loss of activity which could be restored by dithiothreitol. Iodoacetamide, N‐ethylmaleimide and sodium tetrathionate did not inactivate the enzyme. The enzyme was specific for β‐galactosides. Km's for o‐nitrophenyl β‐D‐galactopyranoside and lactose were 2.72 and 13.9 mM, respectively, at pH 6.6 D‐Galactono‐1, 4‐lactone was a good competitive inhibitor (Ki=0.17 mM). pH optimum for hydrolysis of o‐nitrophenyl β‐D‐galactopyranoside was 6.2–6.4. Vmax for this substrate was dependent on two ionizable groups of pKa of 6.13 and 6.51 while Vmax/Km was dependent on two ionizable groups of pKa of 6.39 and 7.23. Activation energy for hydrolysis of o‐nitrophenyl β‐D‐galactopyranoside at pH 7.0 was 9.1 kcal/mol in the range 20–40°C.

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Section: Effect Of Ph On Stabilitymentioning

confidence: 99%

Stability and Enzymatic Properties of ?-Galactosidase From Kluyveromyces Fragilis

Mahoney

Whitaker

1978

J Food Biochemistry

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“…β-Gal is a tetramer composed of four identical polypeptide monomers. Even though each 1,023-amino acid monomer contains one active site for binding and hydrolysis of substrates, assembly of four monomers into a tetramer is required for enzyme activity (2,4). Studies have shown that the interface between each monomer in the tetrameric β-gal is necessary and that the dissociation of the tetramer into dimers or monomers can shut down the enzyme activity completely (5,6).…”

mentioning

confidence: 99%

Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

Jiang

Chong

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.

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“…Escherichia coli ␤-galactosidase is a large 464-kDa tetrameric enzyme of four identical subunits that has been extensively characterized (14,15). ␤-galactosidase is active only as a tetramer (16) and catalyzes the hydrolysis of lactose and other ␤-D-galactopyranosides (17). ␤-galactosidase was the first enzyme to be used for singlemolecule kinetic experiments (18).…”

mentioning

confidence: 99%

Stochastic inhibitor release and binding from single-enzyme molecules

Gorris

Rissin

Walt

2007

Proc. Natl. Acad. Sci. U.S.A.

115

133

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Inhibition kinetics of single-␤-galactosidase molecules with the slow-binding inhibitor D-galactal have been characterized by segregating individual enzyme molecules in an array of 50,000 ultrasmall reaction containers and observing substrate turnover changes with fluorescence microscopy. Inhibited and active states of ␤-galactosidase could be clearly distinguished, and the large array size provided very good statistics. With a pre-steady-state experiment, we demonstrated the stochastic character of inhibitor release, which obeys first-order kinetics. Under steady-state conditions, the quantitative detection of substrate turnover changes over long time periods revealed repeated inhibitor binding and release events, which are accompanied by conformational changes of the enzyme's catalytic site. We proved that the rate constants of inhibitor release and binding derived from stochastic changes in the substrate turnover are consistent with bulk-reaction kinetics.␤-galactosidase ͉ enzyme kinetics ͉ fluorescence microscopy ͉ single molecule T he emergence of new assays for studying enzymes at the single-molecule level has profoundly extended our view of enzyme mechanisms. Whereas stochastic molecular behaviors are hidden by using bulk methods, single-molecule experiments have revealed that, in a population of enzymes, such as lactate dehydrogenase (1), phosphatase (2), or ␤-galactosidase (3), the catalytic rates of individual enzyme molecules are heterogeneous and do not interconvert quickly. Furthermore, it has been shown that the substrate turnover of individual ␤-galactosidase (4), cholesterol oxidase (5), or lipase (6) molecules undergoes dynamic fluctuations in sequential catalytic cycles. These two variations in enzyme activity are referred to as static and dynamic heterogeneity, respectively, and have both been attributed to different conformational states of the enzyme. Despite such heterogeneity, the Michaelis-Menten model derived from bulk enzyme experiments still holds for single-molecule experiments once a stochastic perspective is adopted (4,7,8).Traditionally, one of the most important tools to elucidate an enzyme's catalytic mechanism has been to study the enzyme in the presence of inhibitors. Here, we set out to correlate inhibition mechanisms established in bulk enzyme studies with singleenzyme molecule experiments. To date, inhibitors have been used in single-molecule studies to obtain information about conformational dynamics. Ha et al. (9) showed that it is possible to distinguish between free and inhibitor-bound states of a single-staphylococcal nuclease enzyme molecule. The binding of an inhibitor imposed a conformational constraint on the enzyme molecule resulting in a change of single-molecule polarization and intramolecular single-pair FRET. In this article, we report the direct observation of inhibitor release and binding from single-enzyme molecules by monitoring their substrate turnover.

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Purification and characterization of the multiple forms of β-galactosidase of Escherichia coli

Cited by 59 publications

References 15 publications

Stability and Enzymatic Properties of ?-Galactosidase From Kluyveromyces Fragilis

Stability and Enzymatic Properties of ?-Galactosidase From Kluyveromyces Fragilis

Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

Stochastic inhibitor release and binding from single-enzyme molecules

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