ROLES OF THE AROMATIC RESIDUES CONSERVED IN THE ACTIVE-CENTER OF SACCHAROMYCOPSIS ALPHA-AMYLASE FOR TRANSGLYCOSYLATION AND HYDROLYSIS ACTIVITY

The molecular structure of Saccharomycopsis fibuligera alpha-amylase w as predicted by a homology-based modeling technique, and the amino aci d residues composing the active site were displayed with color codes a ccording to their order of conservation. We noticed two highly conserv ed aromatic residues located in the active center, tyrosine 83 (Y83) a nd tryptophan 84 (W84), and examined their roles in catalytic activity by site-directed mutagenesis. The W, leucine (L), and asparagine (N) mutants at Y83 and the L mutant at W84 showed remarkable enhancement o f transglycosylation activity and complementary decreases in native hy drolysis activity. The phenylalanine (F) mutant at Y83 and the F and Y mutants at W84 only decreased hydrolysis activity. Mechanistic and ki netic studies of these mutants using a reducing-end-blocked substrate and a hydrolysis-specific substrate revealed a probable transglycosyla tion mechanism and critical contributions of the 83rd and 84th aromati c residues to efficient hydrolysis. Given that aromatic residues stack against the faces of sugars, we assumed that Y83 and, presumably, W84 play roles in the binding of oligosaccharide substrates through the s tacking interaction and in the indirect fixation of the catalytic wate r molecule through hydrogen bonding with the hydroxyl of the bound sub strates. Mutations to nonaromatic residues could cause slight changes in the binding topology of substrates to favor transglycosylation over hydrolysis.