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 Escheri chia coli under the control of the lac repressor and promoter have bee n constructed and inserted into Salmonella typhimurium CH3. This syste m expressed the large subunit of the ebg degrees and ebg(a) beta-galac tosidase in high yield (20-60 % of total protein). The large subunits have been purified to homogeneity. As isolated they are tetramers of s ignificant catalytic activity; the N-terminal amino acid residue is Me t, but is not formylated. The k(cat) values fro a series of aryl galac tosides were 6-200-fold reduced from the corresponding values for the holoenzymes. k(cat)/K-m Values for glycosides of acidic aglycones, tho ugh, 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 a nd ebg(a) large subunits were essentially invariant with aglycone pK, suggesting that hydrolysis of the glycosyl-enzyme intermediate had bec ome 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 t he small subunit was thus estimated to cause a 200-fold decrease in de galactosylation rate for ebg degrees and a 20-fold one fro ebg(a). bet a(1g)(V/K) values -0.57 +/- 0.08 for ebg degrees and -0.54 +/- 0.08 fo r ebg(a) isolated subunits were significantly more negative than for h oloenzymes. It is suggested that the small subunit is associated with the optimal positioning of the electrophilic Mg2+ ions in these enzyme s. Use of PCR in the construction of the plasmid also inadvertently le d to the production of psi ebg degrees large subunit in which there wa s a PCR-introduced Leu(9) --> His change. Values of k(cat) for aryl ga lactosides, calculated on the assumption that the psi ebg degrees larg e subunit, like the ebg degrees and ebg(a) large subunits, was 100% ac tive as isolated, were about an order of magnitude lower than for true ebg degrees large subunit, whilst K-m values were similar. The very s ignificant kinetic effect of this inadvertant site-undirected muta-gen esis indicates that quite large kinetic effects of amino-acid replacem ents in enzymes may have no obvious mechanistic significance.