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Biochemical, Molecular, and Genetic Mechanisms

Luminal Starch Substrate "Brake" on Maltase-Glucoamylase Activity Is Located within the Glucoamylase Subunit^1–3,

⁴ CIEP-Facultad de Ciencias Quimicas, Universidad Autonoma de San Luis Potosi, Zona Universitaria, San Luis Potosi, S.L.P., Mexico, 78360; ⁵ USDA, Agricultural Research Service, Children's Nutrition Research Center and Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030-2600; ⁶ Department of Medical Biophysics, University of Toronto and Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, Toronto, M5G1L7 Canada; ⁷ Whistler Center for Carbohydrate Research and Department of Food Science, Purdue University, West Lafayette, IN 47907-2009; ⁸ Division of Biological Sciences, Section of Physiology, Cornell University, Ithaca, NY 14853; ⁹ Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver V6T 1Z3, Canada; and ¹⁰ Institute of Biochemistry and Molecular Medicine, University of Berne, Berne, CH-3012 Switzerland

^* To whom correspondence should be addressed. E-mail: bnichols{at}bcm.tmc.edu.

The detailed mechanistic aspects for the final starch digestion process leading to effective -glucogenesis by the 2 mucosal -glucosidases, human sucrase-isomaltase complex (SI) and human maltase-glucoamylase (MGAM), are poorly understood. This is due to the structural complexity and vast variety of starches and their intermediate digestion products, the poorly understood enzyme-substrate interactions occurring during the digestive process, and the limited knowledge of the structure-function properties of SI and MGAM. Here we analyzed the basic catalytic properties of the N-terminal subunit of MGAM (ntMGAM) on the hydrolysis of glucan substrates and compared it with those of human native MGAM isolated by immunochemical methods. In relation to native MGAM, ntMGAM displayed slower activity against maltose to maltopentose (G5) series glucose oligomers, as well as maltodextrins and -limit dextrins, and failed to show the strong substrate inhibitory "brake" effect caused by maltotriose, maltotetrose, and G5 on the native enzyme. In addition, the inhibitory constant for acarbose was 2 orders of magnitude higher for ntMGAM than for native MGAM, suggesting lower affinity and/or fewer binding configurations of the active site in the recombinant enzyme. The results strongly suggested that the C-terminal subunit of MGAM has a greater catalytic efficiency due to a higher affinity for glucan substrates and larger number of binding configurations to its active site. Our results show for the first time, to our knowledge, that the C-terminal subunit of MGAM is responsible for the MGAM peptide's "glucoamylase" activity and is the location of the substrate inhibitory brake. In contrast, the membrane-bound ntMGAM subunit contains the poorly inhibitable "maltase" activity of the internally duplicated enzyme.