A quantitation of the factors which affect the hydrolase and transgalactosylase activities of β-galactosidase (E. coli) on lactose

Abstract

A study was implemented to quantitate the hydrolase and transgalactosylase activities of beta-galactosidase (E. coli) with lactose as the substrate and to investigate various factors which affect these activities. At low lactose concentrations the rate of galactose production was equal to the rate of glucose production. The rate of galactose production relative to glucose, however, dropped dramatically at lactose concentrations higher than 0.05 M and production of trisaccharides and tetrasaccharides began (galactose/glucose ratios of about 2:1 and 3:1, respectively, were found for these two types of oligosaccharides). At least five different trissacharides were formed and their patterns of formation showed that they probably utilized both lactose and allolactose as galactosyl acceptors. Allolactose was produced in amounts proportional to glucose at all lactose concentrations (ratios of allolactose/glucose were about 0.88). Analyses of various data, including a reaction analyzed at very early times, showed that the major means of production of allolactose (and the only means initially) was the direct enzymatic transfer of galactose from the 4 position to the 6 position of the glucose moiety of lactose without prior release of glucose from the enzyme. It was shown, however, that allolactose could also be formed in significant quantities by the transfer of galactose to the 6 position of free glucose, and also by hydrolysis of preformed trisaccharide. A mechanism which fits the initial velocity data was proposed in which the steps involving the formation of an enzyme-gallactose-glucose complex, the formation and breakage of allolactose on the enzyme, and the release of glucose all seem to be of roughly equal magnitude and rate determining. Various factors affected the amounts of transgalactosylase and hydrolase activities occurring. At high pH values (greater than 7.8) the transgalactosylase/hydrolyase activity ratio increased dramatically while it decreased at low pH values (less than 6.0). At mid pH values the ratio was essentially constant. The absence of Mg2+ caused a large decrease in the transgalactosylase/hydrolase activity ratio while the absence of all but traces of Na+ or K+ had no effect. The anomeric configuration of lactose altered the transgalactosylase/hydrolase activity ratios, alpha-Lactose resulted in a decrease of allolactose production (transgalactosylase activity) relative to hydrolase activities (glucose production) while beta-lactose had the opposite effect.

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

“…As these two substrates have different rate-determining consequences, it is useful to study the kinetics of both in mechanistic studies. Assays of b-galactosidase reactions with lactose, best done with gas-liquid chromatography, 2 are not routinely performed because the assay is technically cumbersome, requires scrutiny of the production of galactose, glucose, and allolactose, and must also account for the effects of these products as transgalactosidic acceptors and for allolactose being a substrate. Even lactose itself is a transgalactosidic acceptor.…”

“…1). 2 First, it can cleave the disaccharide lactose to form glucose and galactose, which can then enter glycolysis. Second, the enzyme can catalyze the transgalactosylation of lactose to allolactose, and, third, the allolactose can be cleaved to the monosaccharides.…”

Isoraffinose (6G-β-galactosylsucrose) Synthesized by the Intermolecular Transgalactosylation Reaction ofEscherichia coliβ-Galactosidase

“…And in the lactosesucrose system, glucose and traces of galactose and fructose were found as monosaccharides, and both allolactose and isoraffinose accumulated during the transgalactosylation. 7 ) showed that the main reaction products at iow initial concentrations of lactose were the hydrolyzates, galactose and glucose (53%), and the intramolecular transgalactosylation reaction product, allolactose (43%), and the accumulation of trisaccharides, so called galactosyl-Iactose, was very low.…”

“…With free glucose as an acceptor, Huber et al 7 ) showed that there was significantly more allolactose produced than when no glucose was present. This indicates that not only glucose but also sucrose can substitute easily for bound glucose (from lactose which is being acted on by p-galactosidase).…”

“…When the natural substrate, lactose is acted upon by the E. coli p-galactosidase, Hurber et a1. 7 ) showed that approximately 50% of the molecules are converted by intramolecular transgalactosylation to allolactose (O-p-D-galactopyranosyl-(1-46)-D-glucose: independent of the initial lactose concentration), while about 50% is either hydrolytic products or intermolecular transgalactosylation products (depending on the initial lactose concentration). In the presence of sugar or alcohol as an acceptor for the intermolecular.…”