Biochemical and kinetic analysis of the GH3 family beta-xylosidase from Aspergillus awamori X-100.
The beta-xylosidase from Aspergillus awamori X-100 belonging to the family 3 glycoside hydrolase revealed a distinctive transglycosylating ability to produce xylooligosaccharides with degree of polymerization more than 7. In order to explain this fact, the enzyme has been subjected to the detailed biochemical study. The enzymatic hydrolysis of p-nitrophenyl beta-D-xylopyranoside was found to occur with overall retention of substrate anomeric configuration suggesting cleavage of xylosidic bonds through a double-displacement mechanism. Kinetic study with aryl beta-xylopyranosides substrates, in which leaving group pK(a)s were in the range of 3.96-10.32, revealed monotonic function of log(k(cat)) and no correlation of log(k(cat)/Km) versus pKa values indicating deglycosylation as a rate-limiting step for the enzymatic hydrolysis. The classical bell-shaped pH dependence of k(cat)/Km indicated two ionizable groups in the beta-xylosidase active site with apparent pKa values of 2.2 and 6.4. The kinetic parameters of hydrolysis, Km and k(cat), of p-nitrophenyl beta-D-1,4-xylooligosaccharides were very close to those for hydrolysis of p-nitrophenyl-beta-D-xylopyranoside. Increase of p-nitrophenyl-beta-D-xylopyranoside concentration up to 80 mM led to increasing of the reaction velocity resulting in k(cat)(app)=81 s(-1). Addition of alpha-methyl D-xylopyranoside to the reaction mixture at high concentration of p-nitrophenyl-beta-D-xylopyranoside (50 mM) caused an acceleration of the beta-xylosidase-catalyzed reactions and appearance of a new transglycosylation product, alpha-methyl D-xylopyranosyl-1,4-beta-D-xylopyranoside, that was identified by 1H NMR spectroscopy. The kinetic model suggested for the enzymatic reaction was consistent with the results obtained.
Eneyskaya
Elena V
EV
Petersburg Nuclear Physics Institute, Russian Academy of Science, Molecular and Radiation Biology Division, Gatchina 188300, Russia.
Ivanen
Dina R
DR
Bobrov
Kirill S
KS
Isaeva-Ivanova
Lyudmila S
LS
Shabalin
Konstantin A
KA
Savel'ev
Andrew N
AN
Golubev
Alexander M
AM
Kulminskaya
Anna A
AA
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D005656:Fungal Proteins / Q000737:chemistry*; D006027:Glycosides / Q000737:chemistry; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000737:chemistry*; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry*
D005656:Fungal Proteins; D006027:Glycosides; D009844:Oligosaccharides; C008646:4-nitrophenyl beta-D-xyloside; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase