Structure-function analysis of human sucrase-isomaltase identifies key residues required for catalytic activity

Abstract

Sucrase-isomaltase (SI) is an intestinal membrane-associated α-glucosidase that breaks down di- and oligosaccharides to absorbable monosaccharides. SI has two homologous functional subunits (sucrase and isomaltase) that both belong to the glycoside hydrolase family 31 (GH31) and differ in substrate specificity. All GH31 enzymes share a consensus sequence harboring an aspartic acid residue as a catalytic nucleophile. Moreover, crystallographic structural analysis of isomaltase predicts that another aspartic acid residue functions as a proton donor in hydrolysis. Here, we mutagenized the predicted proton donor residues and the nucleophilic catalyst residues in each SI subunit. We expressed these SI variants in COS-1 cells and analyzed their structural, transport, and functional characteristics. All of the mutants revealed expression levels and maturation rates comparable with those of the wild-type species and the corresponding nonmutated subunits were functionally active. Thereby we determined rate and substrate specificity for each single subunit without influence from the other subunit. This approach provides a model for functional analysis of the single subunits within a multidomain protein, achieved without the necessity to express the individual subunits separately. Of note, we also found that glucose product inhibition regulates the activities of both SI subunits. We experimentally confirmed the catalytic function of the predicted proton donor residues, and sequence analysis suggested that these residues are located in a consensus region in many GH31 family members. In summary, these findings reveal the kinetic features specific for each human SI subunit and demonstrate that the activities of these subunits are regulated via product inhibition.

Keywords: Glycoside hydrolase family 31; carbohydrate metabolism; enzyme kinetics; glycosidase; product inhibition; site-directed mutagenesis; starch digestion; substrate specificity; sucrase-isomaltase.

Figure 1.

Position and mechanism of action of the catalytic aspartic acid residues in the three-dimensional structure of sucrase-isomaltase protein. A, comparative modeling of the three-dimensional structure of SUC subunit was performed by MODELLER based on its homology to glucoamylase subunit of intestinal maltase-glucoamylase protein (PDB code 3TOP). IM (PDB code 3LPO) and SUC structures were visualized and aligned by UCSF Chimera software. Nucleophilic (left) and proton donor (acid catalyst; right) aspartic acid residues are shown as balls and sticks and highlighted in color. Substrate binding residues as already determined for IM are illustrated by yellow sticks. B, mechanism of maltose hydrolysis by sucrase-isomaltase based on classical Koshland retaining mechanism for α-glucosidases.

Figure 2.

Localization of catalytic aspartic acid residues within SI and overview of generated mutants. A, schematic presentation of the SI protein. The positions of aspartic acid catalytic residues are depicted by arrows, and exchanged amino acid residues are listed. B, consistency of sucrase and isomaltase sequences in the vicinity of their catalytic residues. Sequence alignment of the SI subunits was performed by Profile ALIgNEment (PRALINE).

Figure 3.

Biosynthesis of the SI catalytic site mutants. COS-1 cells transiently expressing either wild type or an active site mutant of SI were lysed, SI was immunoprecipitated and detected by immunoblotting after treatment with endoglycosidase H. The relative amounts of SI_c and SI_h glycoforms in the mutants, as well as the wild-type SI, were quantified and graphically presented as the ratio of total SI. SI_c, complex N- and O-glycosylated mature SI; SI_h, mannose-rich immature form of SI.

Figure 4.

Tryptic structural analysis to determine the folding of SI catalytic site variants. Immunoprecipitants of wild-type SI or its variants prepared from transiently expressing COS-1 cells were subjected to 500 BAEE units of trypsin and detected by Western blotting in comparison with the untreated controls. Immunoblotting (IB) was either performed with the HBB2/614/88 antibody to detect the SUC subunit or the HBB3/705/60 antibody to detect the IM subunit.

Figure 5.

Sucrase, maltase, and palatinase activities of SI active site mutants compared with the wild type. The SI variants were immunoprecipitated from cell lysates of transiently expressing COS-1 cells and assayed for their enzymatic function in hydrolyzing sucrose, maltose, or isomaltulose. The relative specific activities were calculated based on the SI protein content in each sample detected by Western blotting. The relative specific activities of SI normalized to the wild type are presented as means ± S.E. of at least four independent experiments.

Figure 6.

The proton donor aspartic acid occurs in a consensus motif of the GH31 family. A, amino acid sequence of 59 members of GH31 family were aligned by PRALINE multiple sequence alignment toolbox. Sequence consistency for the stretch surrounding the aspartic acid residue that functions as proton donor in human SI subunits is illustrated. B, sequence logo for the consensus motif was generated from the aligned sequences using WebLogo online tool.

Figure 7.

Kinetics of maltase hydrolysis by SI active site mutants in comparison with the wild-type enzyme. COS-1 cells transiently expressing SI-D1500Y, SI-D604Y, or wild-type SI protein were homogenized in phosphate-citrate buffer, pH 6.2, and used for enzyme assay. Kinetics of maltose digestion was determined from each homogenate using different concentration of maltose substrate based on the Michaelis-Menten method. One arbitrary unit (A.U.) is a relative amount of enzyme activity that produces 1 μmol glucose/h.

Figure 8.

Glucose can inhibit the enzyme activity of the SI protein. Michealis-Menten kinetics of the SI protein from brush border membrane preparations of the human small intestine were determined using different concentrations of sucrose or isomaltose as substrate. Here, the presence of 0.7 mm of glucose in the reaction was determined to partly inhibit SI function. Catalytic efficiency was determined as V_max divided by K_m and normalized to the value of the control sample. Each unit is equal to the amount of enzyme activity that liberates 1 μmol glucose/h.