It has been hypothesized that human mucosal glucoamylase (EC 3.2.1.20 and 3.2.1.3) activity serves as an alternate pathway for starch digestion when luminal ␣-amylase activity is reduced because of immaturity or malnutrition and that maltase-glucoamylase plays a unique role in the digestion of malted dietary oligosaccharides used in food manufacturing. As a first step toward the testing of this hypothesis, we have cloned human small intestinal maltase-glucoamylase cDNA to permit study of the individual catalytic and binding sites for maltose and starch enzyme hydrolase activities in subsequent expression experiments. Human maltaseglucoamylase was purified by immunoisolation and partially sequenced. Maltase-glucoamylase cDNA was amplified from human intestinal RNA using degenerate and gene-specific primers with the reverse transcription-polymerase chain reaction. The 6,513-base pair cDNA contains an open reading frame that encodes a 1,857-amino acid protein (molecular mass 209,702 Da). Maltase-glucoamylase has two catalytic sites identical to those of sucrase-isomaltase, but the proteins are only 59% homologous. Both are members of glycosyl hydrolase family 31, which has a variety of substrate specificities. Our findings suggest that divergences in the carbohydrate binding sequences must determine the substrate specificities for the four different enzyme activities that share a conserved catalytic site.Starches are a mixture of two structurally different polysaccharides: amylose, a linear [4-O-␣-D-glucopyranosyl-D-glucose] n polymer, and amylopectin, with additional 6-O-␣-D-glucopyranosyl-D-glucose links (about 4% of total), which result in a branched configuration. Dietary starches are a mixture of approximately 25% amylose in amylopectin, a fact of nutritional significance because of the multienzyme complexity of the mammalian starch digestion pathway (1). ␣-amylase (EC 3.2.1.1) is the endoenzyme found in mature human salivary and pancreatic secretions that produces linear maltose oligosaccharides by hydrolysis of ␣134 linkages of amylose (2, 3). ␣-amylase bypasses the ␣1 3 6 linkages of amylopectin and produces branched isomaltose oligosaccharides. The starchderived oligosaccharides are not fermentable by yeast without further processing by -amylase (EC 3.1.1.2), which hydrolyzes the nonreducing ends at 134 and 136 linkages (2). In mammals, hydrolysis of the nonreducing ends is carried out by small intestinal mucosal brush border-anchored sucrase-isomaltase (SIM) 1 (EC 3.2.1.48 and 3.2.1.10) and maltase-glucoamylase (MGA) (EC 3.2.1.20 and 3.2.1.3) complexes (1). Enzyme substrate specificities of SIM overlap with those of MGA. In vivo, SIM accounts for 80% of maltase (1,4-O-␣-D-glucanohydrolase) activity, all sucrase (D-glucopyranosyl--D-fructohydrolase) activity, and almost all isomaltase (1, 6-O-␣-D-glucanohydrolase) activity (1). MGA accounts for all glucoamylase exoenzyme (1,4-O-␣-D-glucanohydrolase) activity for amylose and amylopectin substrates, 1% of isomaltase activity, and 20% of maltase activity (1...
