SUBSTRATE RECOGNITION AT THE BINDING-SITE IN MAMMALIAN PANCREATIC ALPHA-AMYLASES

Porcine and human pancreatic alpha-amylases (PPA and HPA, respectively) have five binding sites for hexose residues of substrates. As previously reported, when a substrate is too small to occupy subsite 5 of the alpha-amylases, the optimum pH for catalytic activity changes from neutral to acidic [Ishikawa, K., Matsui, I., Honda, K., & Nakatani, H. (1990) Biochemistry 29,7119-7123]. We studied the mechanism by which the enzyme recognizes the substrate by using the synthetic substrates maltopentaose (G5) and its analogs 4-O-alpha-maltotetraosyl-D-xylose (G4-X), 4-O-alpha-maltotetraosyl-2-deoxy-D-glucose (G4-D), 3-O-alpha-maltotetraosyl-L-sorbose (G4-S), 4-O-beta-maltotetraosyl-D-glucose (G4beta-G), alpha-maltotetraosyl-beta-D-fructose (G4-F), p-nitrophenyl alpha-maltotetraoside(G4-phi), and maltopentaitol (G4-GOH). The reducing-end residues of these substrates, relevant to subsite 5, are D-xylose (X), 2-deoxy-D-glucose (D), L-sorbose (S), D-glucose (G), alpha-D-fructofuranose (F), p-nitrophenyl (phi), and D-sorbitol (GOH), respectively. The optimum pH for catalytic activity on the substrates G5, G4-X, G4-D, G4-S, G4beta-G, and G4-phi was neutral, while that for G4-GOH and G4-F was acidic. These results indicate that only six-membered-ring residues and a phenyl group are recognizable by subsite 5. The neutral pH profile for G4-phi suggests that steric compatibility of the substrates at subsite 5 is also important for the enzyme activity. The histidine residue at position 201 is located near subsite 5 and has been shown to be important for substrate recognition in chemical modification studies. Therefore, the catalytic activity of point-mutated HPA (H201N), in which histidine 201 was replaced by asparagine, was examined on the same synthetic substrates. The optimum pH for the mutant H201N HPA was acidic for all the substrates, indicating that subsite 5 of the mutant enzyme did not recognize their reducing-end residues. This indicates that, in addition to steric compatibility, interaction between histidine 201 and the substrate is essential for optimal amylase activity at neutral pH. Furthermore, regulation of the catalytic power and the optimum pH through substrate recognition at subsite 5 was qualitatively explained by an induced-fit effect of a peptide loop containing histidine 201 located at the active site.