Structural explanation for allolactose (lac operon inducer) synthesis by lacZ β-galactosidase and the evolutionary relationship between allolactose synthesis and the lac repressor.

β-Galactosidase (lacZ) has bifunctional activity. It hydrolyzes lactose to galactose and glucose and catalyzes the intramolecular isomerization of lactose to allolactose, the lac operon inducer. β-Galactosidase promotes the isomerization by means of an acceptor site that binds glucose after its cleavage from lactose and thus delays its exit from the site.

Substitution for Asn460 cripples β-galactosidase (Escherichia coli) by increasing substrate affinity and decreasing transition state stability.

Substrate initially binds to β-galactosidase (Escherichia coli) at a 'shallow' site. It then moves ∼3Å to a 'deep' site and the transition state forms. Asn460 interacts in both sites, forming a water bridge interaction with the O3 hydroxyl of the galactosyl moiety in the shallow site and a direct H-bond with the O2 hydroxyl of the transition state in the deep site.

Importance of Arg-599 of β-galactosidase (Escherichia coli) as an anchor for the open conformations of Phe-601 and the active-site loop.

Structural and kinetic data show that Arg-599 of β-galactosidase plays an important role in anchoring the "open" conformations of both Phe-601 and an active-site loop (residues 794-803). When alanine was substituted for Arg-599, the conformations of Phe-601 and the loop shifted towards the "closed" positions because interactions with the guanidinium side chain were lost. Also, Phe-601, the loop, and Na+, which is ligated by the backbone carbonyl of Phe-601, lost structural order, as indicated by large B-factors.

Role of Met-542 as a guide for the conformational changes of Phe-601 that occur during the reaction of β-galactosidase (Escherichia coli).

The Met-542 residue of β-galactosidase is important for the enzyme's activity because it acts as a guide for the movement of the benzyl side chain of Phe-601 between two stable positions. This movement occurs in concert with an important conformational change (open vs. closed) of an active site loop (residues 794-803).

Studies of Glu-416 variants of beta-galactosidase (E. coli) show that the active site Mg(2+) is not important for structure and indicate that the main role of Mg (2+) is to mediate optimization of active site chemistry.

Variants of beta-galactosidase with Valine and with Glutamine replacing Glutamate-416 did not have a Mg(2+) bound at the active site even at high Mg(2+) concentrations (200 mM). They had low catalytic activity and the pH profiles were very different from those of the native enzyme. In addition, substrates, substrate analogs, transition state analogs and galactose bound very poorly.

Direct and indirect roles of His-418 in metal binding and in the activity of beta-galactosidase (E. coli).

The active site of ss-galactosidase (E. coli) contains a Mg(2+) ion ligated by Glu-416, His-418 and Glu-461 plus three water molecules. A Na(+) ion binds nearby.

Practical considerations when using temperature to obtain rate constants and activation thermodynamics of enzymes with two catalytic steps: native and N460T-beta-galactosidase (E. coli) as examples.

The values of the rate constants and the associated enthalpies and entropies of enzymes with two catalytic steps can be measured by determining the effects of temperature on the k (cat) values. Practical considerations that should be taken into account when doing this are presented. The narrow temperature range available with enzymes and the sensitivity of pH to temperature mean that special attention to detail must be taken and this study highlights the assiduousness needed.