Identification of essential ionizable groups and evaluation of subsite affinities in the active site of beta-D-glucosidase F1 from a Streptomyces sp.
Partial amino acid sequences, the essential ionizable groups directly involved in catalytic reaction, and the subsite structure of beta-D-glucosidase purified from a Streptomyces sp. were investigated in order to analyze the reaction mechanism. On the basis of the partial amino acid sequences, the enzyme seemed to belong to the family 1 of beta-glucosidase in the classification of glycosyl hydrolases by Henrissat (1991). Dependence of the V and Km values on pH, when the substrate concentration was sufficiently lower than Km, gave the values of 4.1 and 7.2 for the ionization constants, pKe1 and pKe2 of essential ionizable groups 1 and 2 of the free enzyme, respectively. When the dielectric constant of the reaction mixture was decreased in the presence of 10% methanol, the pKe1 and pKe2, values shifted to higher, to +0.60 and +0.35 pH unit, respectively. The findings supported the notion that the essential ionizable groups of the enzyme were a carboxylate group (-COO-, the group 1) and a carboxyl group (-COOH, the group 2). The subsite affinities Ai's in the active site were evaluated on the basis of the rate parameters of laminarioligosaccharides. Subsites 1 and 2 having positive Ai values (A1 was 1.10 kcal/mol and A2 was 4.98 kcal/mol) were considered to probably facilitate the binding of the substrate to the active site. However, the subsites 3 and 4 showed negative Ai values (A3 was -0.21 kcal/mol and A4 was -2.8 kcal/mol).
Fukuda
Kenji
K
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
Mori
Haruhide
H
Okuyama
Masayuki
M
Kimura
Atsuo
A
Ozaki
Hachiro
H
Yoneyama
Michio
M
Chiba
Seiya
S
D000465:Algorithms; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry* / Q000378:metabolism
D009844:Oligosaccharides; D007294:Inositol; D001617:beta-Glucosidase; C011423:conduritol epoxide