Effect of modifying histidine residues on the action of Bacillus amyloliquefaciens and barley-malt alpha-amylases.
Modification of porcine pancreatic alpha-amylase (PPA) and Taka-amylase A(TAA) with diethyl pyrocarbonate (DEP) causes activation of the release of p-nitrophenol from p-nitrophenol alpha-maltoside (G2PNP), and a decrease in amylase activity (hydrolysis of alpha-1,4 glucosidic bonds in starch). Among the possible sites of modification, attention focuses on three histidine residues present around the active site of alpha-amylases of many different origins. In PPA these are His 101, His 201, and His 299, with His 101 and His 299 being very close to the site of catalysis and thus perhaps directly or indirectly involved in the catalysis. On the other hand, His 201 is located on the aglycon side of the catalytic site, and we have suggested that it is involved in the increase of PNP release after chemical modification. Investigations of site-directed mutagenesis of the histidine residues of human pancreatic alpha-amylase support this identification. Although the degree of sequence similarity among alpha-amylases of different origins is low, there are several conserved short regions. Most belong to the structural components of the active site in PPA and TAA. Furthermore, there is a close similarity in the three-dimensional structures of PPA and TAA. The conserved residues around the active site in all alpha-amylases suggest some universal structural similarities in these active sites. Therefore, we examined the effects of the chemical modification of histidine residues in Bacillus amyloliquefaciens alpha-amylase(BLA) with DEP and made a comparison with modification of barley alpha-amylase isoenzyme II(BAII), using identical substrate systems. These two alpha-amylases have more substrate binding subsites than PPA and TAA, and have similar action patterns with malto-oligosaccharides.
Nakatani
H
H
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
Hamaguchi
K
K
Ishikawa
K
K
D001407:Bacillus / Q000201:enzymology*; D002240:Carbohydrate Sequence; D006639:Histidine; D001467:Hordeum / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009596:Nitrophenols; D009844:Oligosaccharides / Q000378:metabolism*; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000378:metabolism*
D009596:Nitrophenols; D009844:Oligosaccharides; C021705:maltooligosaccharides; D006639:Histidine; D000516:alpha-Amylases; C024836:4-nitrophenol