Shaikh Fathima Aidha, Randriantsoa Mialy, Withers Stephen G
Department of Chemistry, 2036 Main Mall, University of British Columbia, Vancouver V6T 1Z1, Canada.
Biochemistry. 2009 Sep 8;48(35):8396-404. doi: 10.1021/bi900991h.
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.
A抗原和B抗原在输血和器官移植中至关重要。产气荚膜梭菌的一种内切β-半乳糖苷酶EABase对从糖缀合物中裂解A和B三糖的特异性是独特的，在修饰细胞表面血型抗原方面具有巨大的应用潜力。此前尚未通过实验确定该酶及其家族（GH98）中其他酶的作用机制以及其催化残基的身份。通过EABase对合成底物DNP-β-A-三糖水解的直接1H NMR分析表明，EABase是一种转化型内切β-半乳糖苷酶。在pH 6.0、37℃条件下，使用活化和未活化的底物对EABase及其突变体（E354A、D429A、D453A、E467A和E506A）进行动力学表征。EABase对DNP-β-A-三糖的水解遵循正常的米氏动力学，表观KM为64±3μM，k(cat)为105±5 min(-1)。将两个假定的活性位点残基D453和E506突变为丙氨酸导致活性完全丧失，强烈表明这些残基中的一个或两个作为碱催化剂发挥作用。动力学数据还强烈表明E354是酸催化剂，因为E354A突变体对未活化天然底物的活性比野生型酶低1100倍，而用活化的芳基糖苷底物（DNP-β-A-三糖）测定时其活性仅低10倍。通过比较野生型和E354A突变体的pH曲线获得了进一步的支持：酸催化剂的突变消除了野生型酶k(cat)/KM的钟形pH依赖性中的碱性部分。
