Identification of catalytic residues of Ca2+-independent 1,2-alpha-D-mannosidase from Aspergillus saitoi by site-directed mutagenesis.
The roles of six conserved active carboxylic acids in the catalytic mechanism of Aspergillus saitoi 1,2-alpha-d-mannosidase were studied by site-directed mutagenesis and kinetic analyses. We estimate that Glu-124 is a catalytic residue based on the drastic decrease of kcat values of the E124Q and E124D mutant enzyme. Glu-124 may work as an acid catalyst, since the pH dependence of its mutants affected the basic limb. D269N and E411Q were catalytically inactive, while D269E and E411D showed considerable activity. This indicated that the negative charges at these points are essential for the enzymatic activity and that none of these residues can be a base catalyst in the normal sense. Km values of E273D, E414D, and E474D mutants were greatly increased to 17-31-fold wild type enzyme, and the kcat values were decreased, suggesting that each of them is a binding site of the substrate. Ca2+, essential for the mammalian and yeast enzymes, is not required for the enzymatic activity of A. saitoi 1,2-alpha-d-mannosidase. EDTA inhibits the Ca2+-free 1,2-alpha-d-mannosidase as a competitive inhibitor, not as a chelator. We deduce that the Glu-124 residue of A. saitoi 1,2-alpha-d-mannosidase is directly involved in the catalytic mechanism as an acid catalyst, whereas no usual catalytic base is directly involved. Ca2+ is not essential for the activity. The catalytic mechanism of 1,2-alpha-d-mannosidase may deviate from that typical glycosyl hydrolase.
Tatara
Yota
Y
Laboratory of Molecular Enzymology, Graduate School of Engineering, Soka University, Hachioji, Tokyo, 192-8577, Japan.
Lee
Byung Rho
BR
Yoshida
Takashi
T
Takahashi
Koji
K
Ichishima
Eiji
E
D001230:Aspergillus / Q000201:enzymology*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D020134:Catalytic Domain; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008361:Mannosidases / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013696:Temperature; D013997:Time Factors
D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D002118:Calcium