CATALYSIS BY THE LARGE SUBUNIT OF THE 2ND BETA-GALACTOSIDASE OF ESCHERICHIA-COLI IN THE ABSENCE OF THE SMALL-SUBUNIT

Plasmids containing the ebgA degrees and ebgA degrees genes of Escherichia coli under the control of the lac repressor and promoter have been constructed and inserted into Salmonella typhimurium CH3. This system expressed the large subunit of the ebg degrees and ebg(a) 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 is not formylated. The k(cat) values fro a series of aryl galactosides were 6-200-fold reduced from the corresponding values for the holoenzymes. k(cat)/K-m Values for glycosides of acidic aglycones, though, were unchanged, whilst k(cat)/K-m values for galactosides of less acidic aglycones showed a modest (up to 10-fold) decrease. The k(cat) values for glycosides of acidic aglycones hydrolysed by ebg degrees and ebg(a) large subunits were essentially invariant with aglycone pK, suggesting that hydrolysis of the glycosyl-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 ebg degrees and a 20-fold one fro ebg(a). beta(1g)(V/K) values -0.57 +/- 0.08 for ebg degrees and -0.54 +/- 0.08 for ebg(a) 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 ebg degrees large subunit in which there was a PCR-introduced Leu(9) --> His change. Values of k(cat) for aryl galactosides, calculated on the assumption that the psi ebg degrees large subunit, like the ebg degrees and ebg(a) large subunits, was 100% active as isolated, were about an order of magnitude lower than for true ebg degrees large subunit, whilst K-m values were similar. The very significant kinetic effect of this inadvertant site-undirected muta-genesis indicates that quite large kinetic effects of amino-acid replacements in enzymes may have no obvious mechanistic significance.