1991 Fiscal Year Final Research Report Summary
studies on New Reactions in Glycosylase Catalysis
| Project/Area Number |
02806014
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
応用生物化学・栄養化学
|
|---|
| Research Institution | Hokkaido University |
|---|
Principal Investigator |
MATSUI Hirokazu Faculty of Agriculture Lecturer, 農学部, 助手 (90109504)
|
|---|
| Project Period (FY) |
1990 – 1991
|
| Keywords | Glycosylase / alpha-Glucosidase / Galactosidase / Glucoamylase / beta-Glucosyl fluoride / Anomer / Reaction mechanism / Hydration |
|---|
| Research Abstract |
Aspergillus niger alpha-glucosidase catalyzed the slow hydroysis of beta-Dglucopyranosyl fluoride to form alpha-D-glucose. Maltotriose competitively inhibited the hydrolysis, and beta-D-glucosyl fluoride in turn competitively inhibited the hydrolysis of p-nitrophenyl alpha-D-glucopyranoside, indicating that beta-D-glucosyl fluoride is bound at the same site as known substrates for the alpha-glucosidase.
Also alpha-glucosidases from sugar beet and rice catalyzed the hydrolysis of beta-D-glucosyl fluoride to form alpha-D-glucose. The reaction was slow, with V/K 1-2% of that for PNP alpha-D-glucoside hydrolysis, but was not due to any impurity in the substrate or to contaminating beta-glucosidase or glucoamylase. Maltopentaose was a linear competitive inhibitor of PNP alpha-glucoside hydrolysis in each case. Almond beta-glucosidase, promoted alpha-D-glucosyl fluoride hydrolysis to form beta-glucose at an exceedingly low rate. This weak reaction did not stem from any impurity in substance,
… More
or to contamination with alpha-glucosidase or glucoamylase, but it was partly(ca. 20%)attributable to a trace of accompanying trehalase.
That the disfavored D-glucosyl fluoride in each case was converted to a product of the same configuration as from enitols or favored D-glucosyl substrates provides ndw evidence for a separate stage of catalysis in which the steric outcome of reactions of a glucosidase is strictly conserved by protein structure regardless of substrate configuration.
Coffee bean alpha-galactosidase was found to catalyze the hydration of D-galactal and D-galacto-octenitol, each a known substrate for beta-galactosidase. The enzyme protonated each substrate from beneath the plane of the ring, as assumed for alpha-D-galactosides. These results provide an unequivocal assignment of the orientation of an acidic catalytic group to the alpha-galactosidase reaction center. The findings are discussed in light of the concept that catalysis by glycosidases involves a "plastic" protonation phase and a "conserved" product configuration phase. Less
|
