Alteration of bond-cleavage pattern in the hydrolysis catalyzed by Saccharomycopsis alpha-amylase altered by site-directed mutagenesis.
The 210th lysine (K) residue in the Saccharomycopsis alpha-amylase (Sfamy) molecule was replaced by arginine (R) and asparagine (N) residues by site-directed mutagenesis. The influences of the replacements on the bond-cleavage pattern for several substrates were analyzed. Both mutant enzymes, K210R and K210N, cleave mainly the first glycosidic bond from the reducing end of maltotetraose (G4), while the native enzyme hydrolyzes mainly the second bond from the reducing end. We changed successfully the major cleavage point in the hydrolysis reaction of G4. The 8th subsite affinities of the K210R and K210N enzymes are calculated to be +2.52 and -0.01 kcal/mol, respectively, whereas that of the native enzyme is +3.32 kcal/mol as reported in the previous paper. These affinity values suggest that the K210 residue composes the 8th subsite, one of major subsites, and that a positively charged amino residue is necessary for the 8th subsite affinity. The K210N enzyme is found to be less active for short substrates like maltotetraose (G4) than for long substrates like amylose A (approximately G18). The reduced catalytic activity specifically for the short substrates is also attributable to the remarkable decrease in the affinity of the 8th subsite.
Matsui
I
I
National Chemical Laboratory for Industry, Ibaraki, Japan.
Ishikawa
K
K
Miyairi
S
S
Fukui
S
S
Honda
K
K
D000595:Amino Acid Sequence; D000688:Amylose / Q000378:metabolism; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D008320:Maltose / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012442:Saccharomycopsis / Q000201:enzymology*; D013379:Substrate Specificity; D014170:Transformation, Genetic; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
D009844:Oligosaccharides; D008320:Maltose; D000688:Amylose; C009819:maltotetraose; D000516:alpha-Amylases