Substrate recognition at the binding site in mammalian pancreatic .alpha.-amylases

Abstract

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 (G4 beta-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, G4 beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)