Mechanistic analysis of the blood group antigen-cleaving endo-beta-galactosidase from Clostridium perfringens.
The A and B antigens are of vital importance in blood transfusion and organ transplantation. The specificity of EABase, an endo-beta-galactosidase from C. perfringens, toward the cleavage of A and B trisaccharides from glycoconjugates is unique and holds significant potential for use in modifying blood group antigens on cell surfaces. The mechanism of this enzyme and others in its family (GH98) and the identities of its catalytic residues have not previously been experimentally determined. Direct 1H NMR analysis of the hydrolysis of a synthetic substrate, DNP-beta-A-trisaccharide, by EABase revealed that EABase is an inverting endo-beta-galactosidase. Both activated and nonactivated substrates were used to kinetically characterize EABase and its mutants (E354A, D429A, D453A, E467A, and E506A) at pH 6.0, 37 degrees C. Hydrolysis of DNP-beta-A-trisaccharide by EABase follows normal Michaelis-Menten kinetics with an apparent KM of 64 +/- 3 microM and a k(cat) of 105 +/- 5 min(-1). Mutation of two putative active site residues, D453 and E506, to alanine resulted in complete loss of activity, strongly suggesting that one or both of these residues functions as the base catalyst. The kinetic data also strongly suggest that E354 is the acid catalyst since the activity of the E354A mutant with nonactivated natural substrates is 1100-fold lower than that of the wild type enzyme, while its activity is only 10-fold lower when assayed with an activated aryl glycoside substrate (DNP-beta-A-trisaccharide). Further support is obtained through comparison of pH profiles for the wild type and E354A mutants: mutation of the acid catalyst eliminates the basic limb from the bell-shaped pH-dependence of k(cat)/KM seen for the wild type enzyme.
Shaikh
Fathima Aidha
FA
Department of Chemistry, 2036 Main Mall, University of British Columbia, Vancouver V6T 1Z1, Canada.
Randriantsoa
Mialy
M
Withers
Stephen G
SG
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D001789:Blood Group Antigens / Q000097:blood; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D003016:Clostridium perfringens / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed / Q000379:methods; D019906:Nuclear Magnetic Resonance, Biomolecular; D013379:Substrate Specificity / Q000235:genetics; D014312:Trisaccharides / Q000032:analysis*; D001616:beta-Galactosidase / Q000378:metabolism
D001789:Blood Group Antigens; D014312:Trisaccharides; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase; D000409:Alanine; C069225:A-trisaccharide