Catalytic mechanism of inulinase from Arthrobacter sp. S37.
Detailed catalytic roles of the conserved Glu323, Asp460, and Glu519 of Arthrobacter sp. S37 inulinase (EnIA), a member of the glycoside hydrolase family 32, were investigated by site-directed mutagenesis and pH-dependence studies of the enzyme efficiency and homology modeling were carried out for EnIA and for D460E mutant. The enzyme efficiency (k(cat)/K(m)) of the E323A and E519A mutants was significantly lower than that of the wild-type due to a substantial decrease in k(cat), but not due to variations in K(m), consistent with their putative roles as nucleophile and acid/base catalyst, respectively. The D460A mutant was totally inactive, whereas the D460E and D460N mutants were active to some extent, revealing Asp460 as a catalytic residue and demonstrating that the presence of a carboxylate group in this position is a prerequisite for catalysis. The pH-dependence studies indicated that the pK(a) of the acid/base catalyst decreased from 9.2 for the wild-type enzyme to 7.0 for the D460E mutant, implicating Asp460 as the residue that interacts with the acid/base catalyst Glu519 and elevates its pK(a). Homology modeling and molecular dynamics simulation of the wild-type enzyme and the D460E mutant shed light on the structural roles of Glu323, Asp460, and Glu519 in the catalytic activity of the enzyme.
Kim
Kyoung-Yun
KY
School of Agricultural Biotechnology, Seoul National University, San 56-1, Sinlim-Dong, Gwanak-Gu, Seoul 151-742, Republic of Korea.
Nascimento
Alessandro S
AS
Golubev
Alexander M
AM
Polikarpov
Igor
I
Kim
Chung-Sei
CS
Kang
Su-Il
SI
Kim
Su-Il
SI
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001173:Arthrobacter / Q000201:enzymology*; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D017124:Conserved Sequence; D018698:Glutamic Acid / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016415:Sequence Alignment
D001426:Bacterial Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D006026:Glycoside Hydrolases; C023086:inulinase