Catalysis by the large subunit of the second beta-galactosidase of Escherichia coli in the absence of the small subunit.
Plasmids containing the ebgAo and ebgAa genes of Escherichia coli under the control of the lac repressor and promoter have been constructed and inserted into Salmonella typhimurium CH3. This system expresses the large subunit of the ebgo and ebga beta-galactosidase in high yield (20-60% of total protein). The large subunits have been purified to homogeneity. As isolated they are tetramers of significant catalytic activity; the N-terminal amino acid residue is Met, but it is not formylated. The kcat. values for a series of aryl galactosides were 6-200-fold reduced from the corresponding values for the holoenzymes. kcat/Km Values for glycosides of acidic aglycones, though, were unchanged, whilst kcat./Km values for galactosides of less acidic aglycones showed a modest (up to 10-fold) decrease. The kcat. values for glycosides of acidic aglycones hydrolysed by ebgo and ebga large subunits were essentially invariant with aglycone pK, suggesting that hydrolysis of the galactosyl-enzyme intermediate had become rate-determining for these substrates. Rate-determining hydrolysis of the glycosyl-enzyme intermediate was confirmed by pre-steady-state measurements and nucleophilic competition with methanol. Absence of the small subunit was thus estimated to cause a 200-fold decrease in degalactosylation rate for ebgo and a 20-fold one for ebga. beta 1g(V/K) values of -0.57 +/- 0.08 for ebgo and -0.54 +/- 0.08 for ebga isolated subunits were significantly more negative than for holoenzymes. It is suggested that the small subunit is associated with the optimal positioning of the electrophilic Mg2+ ions in these enzymes. Use of PCR in the construction of the plasmid also inadvertently led to the production of psi ebgo large subunit in which there was a PCR-introduced Leu9-->His change. Values of kcat. for aryl galactosides, calculated on the assumption that the psi ebgo large subunit, like the ebgo and ebga large subunits, was 100% active as isolated, were about an order of magnitude lower than for true ebgo large subunit, whilst Km values were similar. The very significant kinetic effect of this inadvertant site-undirected mutagenesis indicates that quite large kinetic effects of amino-acid replacements in enzymes may have no obvious mechanistic significance.
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004146:Dioxanes; D004926:Escherichia coli / Q000201:enzymology*; D005697:Galactosides / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D000432:Methanol; D008969:Molecular Sequence Data; D009598:Nitrophenylgalactosides / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins; D017421:Sequence Analysis; D012997:Solvents; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004146:Dioxanes; D005697:Galactosides; D011994:Recombinant Proteins; D012997:Solvents; D009598:Nitrophenylgalactosides; D001616:beta-Galactosidase; C025223:1,4-dioxane; D000432:Methanol