Catalysis by the large subunit of the second β-galactosidase of Escherichia coli in the absence of the small subunit

Abstract

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.

Larger increases in sensitivity to paracatalytic inactivation than in catalytic competence during experimental evolution of the second β-galactosidase of Escherichia coli

“…The inactivation rates in the absence of the small subunit in the two cases examined (ebg o and ebg a ) show modest decreases over the inactivation rates of the corresponding intact enzyme. This is expected in the light of the evidence that we presented [5] that indicated that the main function of the small subunit was to alter the active-site conformation such that application of electrophilic catalysis was more effective. (Given the extensive homology between lacZ and ebg enzymes, it is reasonable to assume that a single active site of the ebg enzyme, like that of the lacZ enzyme [1], is composed of residues from two subunits.…”

“…The various ebg enzymes were purified from the sources and by the procedures already described for ebg o , ebg a , ebg b , ebg ab [3,7], ebg abcd and ebg abcde [17], and for the large (α) subunits of ebg a and ebg o [5]. ψebg a and ψebg b are versions of the enzyme with the PCR-induced Leu-9 His mutation, as described previously [5]. Release of 2,4-dinitrophenol from the inactivator by high concentrations of enzyme was monitored at 400 nm.…”

“…Of the 15 amino acid residues that make up the active site of the lacZ enzyme [1], 13 are conserved in the wildtype ebgA gene product. The presence of the ebgC gene product is essential for full catalytic function, despite the active-site residues all belonging to the ebgA gene product ; it appears that the small subunit is essential for the optimal operation of electrophilic catalysis by the active-site Mg# + [5].…”

The crystal structure of Helicobacter pylori HP1029 highlights the functional diversity of the sialic acid‐related DUF386 family

“…). Finally, group VII is characterized by the presence of EbgC, a fourth E. coli paralog that has been described as a component of an enzyme (Ebg) with marginal β‐galactosidase activity . Nonconservative substitutions are observed in EbgC at the metal‐binding site (Fig ), suggesting loss of catalytic activity in this divergent member of the DUF386 family.…”

Experimental evolution of Ebg enzyme provides clues about the evolution of catalysis and to evolutionary potential

“…Michael Sinnott and his colleagues have studied the mechanisms of catalysis, including the transition state structure and constructed free energy profiles, for the wild‐type enzyme (Ebg 0 ), Class I enzyme (Ebg a ), Class II enzyme (Ebg b ), two different Class IV enzymes (Ebg ab and Ebg abcd ) and the Class V enzyme (Ebg abcde ) [17–23].…”