Trp-999 of β-Galactosidase (Escherichia coli) Is a Key Residue for Binding, Catalysis, and Synthesis of Allolactose, the Natural Lac Operon Inducer

Abstract

Trp-999 is a key residue for the action of beta-galactosidases (Escherichia coli). Several site specific substitutions (Phe, Gly, Tyr, Leu) for Trp-999 were made. Each substitution caused greatly decreased affinities for substrates and inhibitors that bind in the "shallow" mode, while the affinities of inhibitors that bind in the "deep" mode were not decreased nearly as much. This shows that Trp-999 is important for binding in the shallow mode. The residue is also very important for binding glucose to galactosyl-beta-galactosidase (as a transgalactosidic acceptor). Substitution greatly diminished the affinity for glucose. Substitutions also changed the activation thermodynamics and, subsequently, the rates of the catalytic reactions. The enthalpies of activation of the glycolytic bond cleavage step (galactosylation, k(2)) became less favorable while the entropies of activation of that step became more favorable as a result of the substitutions. Differing magnitudes of these enthalpic and entropic effects with ONPG as compared to PNPG caused the k(2) values for ONPG to decrease but to increase for PNPG. The enthalpies of activation for the common hydrolytic step (degalactosylation, k(3)) increased while the entropies of activation for this step did not change much. As a result, k(3) became small and rate determining for each substituted enzyme. The substitutions caused the rate constant (k(4)) of the transgalactosidic acceptor reactions with glucose (for the formation of allolactose) to become much larger and of the same order of magnitude as the normally large rate constants for transgalactosidic acceptor reactions with small alcohols. This is probably because glucose can approach with less restriction in the absence of Trp-999. However, since glucose binds very poorly to the galactosyl-beta-galactosidases with substitutions for Trp-999, the proportion of lactose molecules converted to allolactose is small. Thus, Trp-999 is also important for ensuring that an appropriate proportion of lactose is converted to allolactose.

Structural Explanation for Allolactose (lac Operon Inducer) Synthesis by lacZ β-Galactosidase and the Evolutionary Relationship between Allolactose Synthesis and the lac Repressor

“…Previous studies have shown that binding of Glc is very poor upon substituting for Trp-999 (33). Also reported is a K i Љ value of a substitution (N460A-␤-galactosidase, one of several that could have been chosen) that is unlikely to influence Glc binding.…”

“…The structures described above indicated that His-418, Asn-102, and Trp-999 are also important for binding Glc. However, these three residues were not useful for uniquely identifying ␤-galactosidases that synthesize allolactose as they have other important mechanistic roles (8,(33)(34)(35) and are highly conserved among all ␤-galactosidases examined.…”

LacZ β‐galactosidase: Structure and function of an enzyme of historical and molecular biological importance

“…The substrate in the shallow mode takes up a position roughly parallel to Trp999 with the galactosidic oxygen more or less centered over the indole 8,29,64 [ Fig. 5(a)].…”

Modelling of carbohydrate–aromatic interactions: ab initio energetics and force field performance

“…These complexes are characterized by parallel orientation of aromatic ring and pyranose or furanose moiety with C-H bonds of a carbohydrate pointing onto the plane of an aromatic ring. The role of aromatic amino acid residues in binding of carbohydrate ligands was studied in a variety of model proteins by site-directed mutagenesis [18][19][20], covalent modification [18], and by calorimetric methods [20]. Mutation of such aromatic amino acid residue (to alanine or other non-aromatic residues) generally leads to a significant drop in affinity or activity towards a carbohydrate ligand/substrate [18][19][20].…”