The deletion of amino-terminal domain in Thermoactinomyces vulgaris R-47 alpha-amylases: effects of domain N on activity, specificity, stability and dimerization.
Thermoactinomyces vulgaris R-47 alpha-amylases, TVA I and TVA II, have a domain N, which is an extra structure in the family 13 enzymes. To investigate the roles of domain N in TVAs, we constructed TVAs-deltaN mutants which are deleted in domain N, and Y14,16,68A and Y41,82,95A mutants of TVA II. TVAs-deltaN were unstable under alkaline conditions, and their thermal stabilities were 10 degrees C lower than that of wild-types. The specific activities of TVAs-deltaN for pullulan, starch, cyclodextrins, and oligosaccharides were drastically decreased, being about 1,500- to 10,000-fold smaller than those of wild-types. The kcat values of Y14,16,68A and Y41,82,95A for all tested substrates were markedly decreased, and the Km value of Y14,16,68A for alpha-CD and maltotriose were 25- and 3-fold larger, and that of Y41,82,92A for starch was 10-fold larger than that of the wild-type. TVA I and TVAs-deltaN in solution are a monomer, while TVA II is a homo-dimer, calculated by their molecular masses. These results suggest domain N in TVAs is an important structure for stabilization of enzymes, recognition and hydrolysis of substrates, and dimerization of TVA II.
Yokota
T
T
Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Fuchu-shi, Japan.
Tonozuka
T
T
Kamitori
S
S
Sakano
Y
Y
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008848:Micromonosporaceae / Q000201:enzymology* / Q000235:genetics*; D008958:Models, Molecular; D008970:Molecular Weight; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D007527:Isoenzymes; D000516:alpha-Amylases