Mutational analysis of the roles in catalysis and substrate recognition of arginines 54 and 305, aspartic acid 309, and tryptophan 317 located at subsites 1 and 2 in glucoamylase from Aspergillus niger

The mutants Arg54-->Leu, Arg54-->Lys, Arg305-->Lys, Asp309-->Glu, and Trp317-->Phe, located at subsites 1 and 2 in glucoamylase from Aspergillus niger, provide insight into the importance of specific hydrogen bonds and hydrophobic interactions in substrate recognition, catalytic mechanism, and stability. As suggested from the crystal structure of a closely related glucoamylase [Aleshin, A. E., Firsov, L. M., & Honzatko, R. B. (1994) J. Biol. Chem. 269, 15631-15639], Arg54 in subsite 1 hydrogen bonds to the key polar group 4'-OH of maltose. The two mutants of Arg54 display losses in transition-state stabilization of 16-21 kJ mol-1 in the hydrolysis of different maltooligodextrins, which originate from a [(1.2-1.8) x 10(3)]-fold reduction in kcat and changes in Km ranging from 25% to 300% of the wild-type values. Arg305 similarly hydrogen bonds to 2'-OH and 3-OH, located at subsites 1 and 2, respectively. Arg305-->Lys glucoamylase is not saturated at concentrations of maltose or maltoheptaose of 400- and 40-fold, respectively, the Km of the wild-type enzyme. This mutant also has highly reduced kcat. On the other hand, for the alpha-1,6-linked isomaltose, the Lys305 mutant surprisingly has the same Km as the wild-type enzyme, while kcat is 10(3)-fold reduced. Arg305 is thus an important determinant in the distinction of the alpha-1,4 to alpha-1,6 substrate specificity. Arg305 interacts electrostatically and hydrophobically with the side chains of Asp309 and Trp317.