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.(ABSTRACT TRUNCATED AT 250 WORDS)
Frandsen
T P
TP
Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Christensen
T
T
Stoffer
B
B
Lehmbeck
J
J
Dupont
C
C
Honzatko
R B
RB
Svensson
B
B
D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001234:Aspergillus niger / Q000201:enzymology*; D001483:Base Sequence; D002384:Catalysis; D004252:DNA Mutational Analysis; D017931:DNA Primers; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019791:Guanidine; D006146:Guanidines; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013816:Thermodynamics; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D006146:Guanidines; D001224:Aspartic Acid; D014364:Tryptophan; D001120:Arginine; D005087:Glucan 1,4-alpha-Glucosidase; D019791:Guanidine