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 --> G lu, and Trp317 --> Phe, located at subsites 1 and 2 in glucoamylase fr om 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 stru cture 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) a nd changes in K-m ranging from 25% to 300% of the wild-type values. Ar g305 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, respec tively, the K-m of the wild-type enzyme. This mutant also has highly r educed 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 enzy me, while k(cat) is 10(3)-fold reduced. Arg305 is thus an important de terminant in the distinction of the alpha-1,4 to alpha-1,6 substrate s pecificity. Arg305 interacts electrostatically and hydrophobically wit h 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 iso maltose increased 3-7-fold and k(cat) decreased 2-4-fold compared to t hat 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 temp erature and in guanidine hydrochloride. The results provide insight us eful for rational engineering of bond-type specificity in glucoamylase .