Abstract
An α-amylase II (TVA II) produced by Thermoactinomyces vulgaris R-47 exhibits wide substrate specificity for starch, pullulan, cyclodextrins and isopanose. Asp465 and Arg469 are located at subsite (-2) and are observed among many α-amylase family enzymes. Here, we have investigated the relationship between the substrate specificity of TVA II and the substrate binding by Asp465 and Arg469 using site-directed mutagenesis and X-ray crystallographic analyses. The five mutated TVA IIs (D465N, D465E, D465Q, R469L and R469K) showed lower specific activities for all substrates tested here than the wild-type TVA II. In particular, the mutation of Arg469 significantly decreased the hydrolyzing activities compared to the mutation of Asp465. While the Km values for α-cyclodextrin were increased for all the mutants, large decreases of the kcat values were observed only for R469L and R469K. The side chain of Arg469 is close to that of Asp421, which is one of three catalytic residues, and the positive charge of Arg469 seems to affect the catalytic mechanism via Asp421. These findings suggest that Asp465 and Arg469 are important residues for the substrate binding and the catalytic reaction. Furthermore, the binding of the α-maltosyl unit by Asp465 and Arg469 is commonly observed among TVA II and other α-amylase family enzymes, which is thought to be the basic mechanism of substrate binding.
