Substrate specificity of small‐intestinal lactase

Abstract

Lactase-phlorizin hydrolase is a disaccharidase present in the small intestine of mammals. This enzyme has two active sites, one being responsible for the hydrolysis of lactose. Lactase activity is thought to be selective towards glycosides with a hydrophilic aglycon. In this work, we report a systematic study on the importance of each hydroxyl group in the substrate molecule for lactase activity. For this purpose, all of the monodeoxy derivatives of methyl b-lactoside and other lactose analogues are studied as lactase substrates. With respect to the galactose moiety, it is shown here that HO-3' and HO-2' are necessary for hydrolysis of the substrates by lactase. Using these chemically modified substrates, it has been confirmed that lactase does not behave as a typical P-galactosidase, since it does not show an absolute selectivity with respect to substitution and stereochemistry at C4' in the galactose moiety of the substrate. However, the glucose moiety, in particular the HO-6, appears to be important for substrate hydrolysis, although none of the hydroxyl groups seemed to be essential. In order to differentiate both activities of the enzyme, a new assay for the phlorizin-hydrolase activity has also been developed.Small-intestinal disaccharidases in higher animals are located in the luminal membrane of intestinal mucose where they hydrolyze the disaccharides in the diet and the oligosaccharides that arise in the intestinal lumen after a-amiIolysis of starch [l, 21. Lactase is the small-intestinal disaccharidase that splits the major 8-glycoside of the diet, lactose, to give glucose and galactose (Scheme 1). The enzyme has been the subject of considerable investigation because of its involvement in the most frequent genetically based syndrome in man, known as adult-type alactasia or lactose intolerance in the adult resulting in a remarkable decrease in lactase activity in adulthood which affects more than 33% of adult humans [I, 3,4].A biological clock appears to exist, causing a marked decline of lactase after weaning, except for individuals whose ancestors are dependent on a substantial consumption of milk and milk-derived products.It has been known for some time that, in addition to lactase activity, the enzyme carries a P-glucosidase activity refered to as phlorizin hydrolase [5-81, which is in fact a glycosylceramidase [9]. The enzyme is therefore named lactasephlorizin hydrolase or the intestinal P-glycosidase complex. We have now carried out a systematic study of the substrate specificity with respect to the lactase site of the bglycosidase complex from sheep small intestine, using chemically modified methyl P-lactoside derivatives. Methyl P-lactoside rather than lactose, has been used as a reference compound in order to simplify the analysis of the reaction mixtures. These chcmically modified substrates have been frequently used in the study of enzyme mechanisms [13 -161 and have proved extremely useful in probing the combining sites for the elucidation of specific interactions between proteins and carb...

Noninvasive Evaluation of Intestinal Lactase with 4-Galactosylxylose: Comparison with 3- and 2-Galactosylxylose and Optimization of the Method in Rats

“…Paradoxically, it is the worst substrate that turned out to be most suitable to detect the changes in lactase activity in vivo with high reliability. These results agree with previous reports on the role of each hydroxyl group of the lactose molecule in its interaction with intestinal lactase isolated from lambs (26,27 ). We obtained evidence that the rate-limiting step in the overall absorption of d-xylose from 4-galactosylxylose is not its transport but the hydrolysis of the disaccharide by lactase, and that this compound does not permeate the intestinal mucosa, as reported for lactose (1,28 ).…”

Enzymatic Hydrolysis of Pyridoxine-5′-β-d-glucoside Is Catalyzed by Intestinal Lactase-Phlorizin Hydrolase

“…This recognition was sequence-specific, since the pre-immune serum and pep- tide-competed antibody did not detect the high molecular mass bands that were recognized by the whole immune serum. Values of K m and V max (lactose) calculated for purified rat LPH were consistent with published values (27,34) (Table I). The kinetic parameters (for lactose and PNG) appeared to be representative of the concentrations of substrates that would be encountered physiologically by LPH.…”

“…Glucose release was quantified using a reagent kit based on the glucose oxidase reaction with colorimetric detection. Phlorizin hydrolase activity was measured by reverse-phase HPLC with UV detection of the aglycone, phloretin (27). Standard assay mixtures contained 100 mM phlorizin in 50 mM sodium citrate, pH 5.9.…”

The Biochemistry, Chemistry and Physiology of the Isoflavones in Soybeans and their Food Products

“…21 The latter, lactase-phlorizin hydrolase, is also responsible for lactose hydrolysis. 22 Interestingly, its absence in many Asians may mean that the isoflavone b-glucosides are not readily hydrolyzed in the small intestine. The 6''-O-malonyl-or 6''-O-acetyl-7-O-b-glucosides are poorly hydrolyzed by b-glucosidases and therefore enter the large bowel where the microorganism concentrations are much higher.…”