Alteration of bond-cleavage pattern in the hydrolysis catalyzed by Saccharomycopsis .alpha.-amylase altered by site-directed mutagenesis
- 9 June 1992
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 31 (22), 5232-5236
- https://doi.org/10.1021/bi00137a019
Abstract
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.Keywords
This publication has 18 references indexed in Scilit:
- An increase in the transglycosylation activity of Saccharomycopsis α-amylase altered by site-directed mutagenesisBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1991
- Expression, glycosylation and secretion of fungal hydrolases in yeastTrends in Biotechnology, 1987
- Nucleotide sequence of the α‐amylase gene (ALP1) in the yeast Saccharomycopsis fibuligeraFEBS Letters, 1987
- Purification and some properties of the extracellular α-amylase from Aspergillus awamoriCanadian Journal of Microbiology, 1985
- Effect of surfactants on cyclization of Bacillus macerans cyclodextrin glucanotransferase.Journal of the Japanese Society of Starch Science, 1983
- “Western Blotting”: Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein AAnalytical Biochemistry, 1981
- Subsite affinities of glucoamylase: Examination of the validity of the subsite theoryBiochimica et Biophysica Acta (BBA) - Enzymology, 1973
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970
- Interpretation of dependency of rate parameters on the degree of polymerization of substrate in enzyme-catalyzed reactions. Evaluation of subsite affinities of exo-enzymeBiochemical and Biophysical Research Communications, 1970
- Kinetics of Hydrolytic Reaction Catalyzed by Crystalline Bacterial α-Amylase. III. The Influence of TemperatureBulletin of the Chemical Society of Japan, 1963