MUTATIONAL ANALYSIS OF THE ROLES IN CATALYSIS AND SUBSTRATE RECOGNITION OF ARGININE-54 AND ARGININE-305, ASPARTIC-ACID-309, AND TRYPTOPHAN-317 LOCATED AT SUBSITE-1 AND SUBSITE-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 k(cat) and changes in K-m 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 K-m of the wild-type enzyme. This mutant also has highly reduced k(cat). On the other hand, for the alpha-1,6-linked isomaltose, the Lys305 mutant surprisingly has the same K-m as the wild-type enzyme, while k(cat) 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. The mutants Asp309 --> Glu and Trp317 --> Phe display a 12-80-fold increase in K-m for alpha-1 beta-linked substrates, while k(cat) is essentially unaffected; K-m for isomaltose increased 3-7-fold and k(cat) decreased 2-4-fold compared to that for wild-type glucoamylase. Arg305, Asp309, and Trp317 are near an extraordinary secondary structure [Aleshin, A., Golubev, A., Firsov, L. M., and Honzatko, R. B. (1992) J. Biol. Chem. 267, 19291-19298] in which strain imposed upon Asn313 and Ser411 via hydrogen bonds forces nonbonded atoms in close contact. The Arg305 --> Lys, Asp309 --> Glu, or Trp317 --> Phe mutants thus have reduced stability at elevated temperature and in guanidine hydrochloride. The results provide insight useful for rational engineering of bond-type specificity in glucoamylase.