The determination of subsite binding energies of porcine pancreatic alpha-amylase by comparing hydrolytic activity towards substrates.
The active centre of porcine pancreatic alpha-amylase contains five subsites. Their occupancy has been studied using as a substrate maltooligosaccharide of various chain lengths (maltose up to maltoheptaose), some of their p- and o-nitrophenylated derivatives, and 412-residue amylose. Quantitative analysis of the digestion products allowed the determination of the subsite occupancy for the various productive complexes, the bond cleavage frequency and respective kcati (where i is the binding mode). The catalytic efficiency (kcat/Km) increases with chain length from maltose (2 M-1 X S-1) up to amylose (1.06 X 10(7) M-1 X S-1). The kinetic parameters of p-nitrophenylmaltoside hydrolysis are quite close to those of maltose, and the ortho compound behaves as maltotriose. Determination of binding energy of glucose residue at the various subsites calculated according to the method of Hiromi et al. (Hiromi, K., Nitta, Y., Numata, C. and Ono, S. (1973) Biochim. Biophys. Acta 302, 362-375) did not give consistent results. A method is proposed based on certain properties of porcine pancreatic alpha-amylase, especially the non-interaction of the p-nitrophenyl moiety of the maltose derivative with subsites 1 and 2, and the o-nitrophenyl group which interacts in a similar way to a glucose residue at the reducing end, and on the grounds that the amylase-amylose complexes are of the productive type. In addition, binding energy differences were calculated from substrates with the same chain length. The subsite energy profile is characterized by a low value at subsite 3 which confirms this subsite as the catalytic one. Another consequence is that the hydrolysis rate constant of productive complexes (kintn) (where n is the number of glucose or glucose equivalent residues for a given substrate) varies with chain length which is in conflict with the hypothesis of Hiromi et al.
D000688:Amylose / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004734:Energy Metabolism; D006868:Hydrolysis; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology; D011485:Protein Binding; D013379:Substrate Specificity; D013552:Swine; D000516:alpha-Amylases / Q000378:metabolism*
D009844:Oligosaccharides; D008320:Maltose; D000688:Amylose; D000516:alpha-Amylases