New substrate specificity of modified porcine pancreatic alpha-amylase.
Conversion of the substrate specificity of porcine pancreatic alpha-amylase (PPA) was studied using chemical modification of His residues. Diethyl pyrocarbonate modified His residues in PPA and the activity of the modified PPA for the hydrolysis of the alpha-D-(1,4)glucoside bond in starch or oligosaccharides decreased to less than 1% of that of the native enzyme. However, the activity for the hydrolysis of the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides was increased by chemical modification. When the modified PPA was incubated with a proteinaceous alpha-amylase inhibitor (Mr 60,000) purified from white kidney bean (Phaseolus vulgaris), it bound to the inhibitor. As a result, the remaining less than 1% hydrolytic activity of the modified PPA for starch disappeared completely but that for p-nitrophenyl oligosaccharides remained unaltered. The hydrolytic activity of the native PPA for the alpha-D-(1,4)glucoside bond in oligosaccharides was stronger than that between p-nitrophenyl and oligosaccharides in p-nitrophenyl oligosaccharides. Therefore, when p-nitrophenyl oligosaccharides (three to five glucose residues) were used as substrates for the native PPA, the alpha-D-(1,4)glucoside bonds in the oligosaccharides were hydrolyzed. However, the modified PPA-inhibitor complex hydrolyzed only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides. The above results reveal that, by chemical modification with diethyl pyrocarbonate and biochemical modification with an amylase inhibitor, amylase can be converted to a new exo-type enzyme which hydrolyzes only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides.
Ishikawa
K
K
National Chemical Laboratory for Industry, Ibaraki, Japan.
Hirata
H
H
D000681:Amylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D006639:Histidine; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D037121:Plant Lectins; D010940:Plant Proteins / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine; D013816:Thermodynamics
D009844:Oligosaccharides; D037121:Plant Lectins; D010940:Plant Proteins; C061510:alpha-amylase inhibitor, Phaseolus vulgaris; D006639:Histidine; D008320:Maltose; D000681:Amylases