Abstract
Hydrolysis of starch or oligosaccharides by mammalian amylases, in general, results in maltose as the leaving group. The active site of these amylases harbors three aromatic residues Trp59, Tyr62, and Tyr151, which provide stacking interactions to the bound glucose moieties. We hypothesized that Tyr151, located at the S2′ subsite, may influence the size of the leaving group. Therefore, using a baculovirus expression system, we generated a mutant Y151M in which the tyrosine at position 151 of human salivary amylase is replaced by a methionine. The specific activity, Km, rate of hydrolysis, and the product distribution for Y151M were distinctly different from those of the wild-type enzyme using starch and oligosaccharides as substrates. The mutant enzyme Y151M consistently produced glucose as the minimal leaving group and exhibited a twofold increase in Km. These results suggest that the stacking interaction at subsite S2′ in the wild type plays a role in hydrolysis.
| Original language | American English |
|---|---|
| Pages (from-to) | 468-473 |
| Number of pages | 6 |
| Journal | Biochemical and Biophysical Research Communications |
| Volume | 292 |
| Issue number | 2 |
| DOIs | |
| State | Published - 2002 |
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology
- Cell Biology
Keywords
- Amylase
- Catalysis
- Enzyme kinetics
- Glucose formation
- HPLC separation
- Oligosaccharides
- Site-directed mutagenesis