The main α-galactosidase was purified to homogeneity, in 30% yield, from a solid culture of Phanerochaete chrysosporium on 1 part wheat bran/2 parts thermomechanical softwood pulp. It is a glycosylated tetramer of 50 kDa peptide chains, which gives the N-terminal sequence ADNGLAITPQMG(?W)NT(?W)NHFG(?W)DIS(?W)DTI. It is remarkably stable, with crude extracts losing no activity over 3 h at 80 °C, and the purified enzyme retaining its activity over several months at 4 °C. The kinetics of hydrolysis at 25 °C of various substrates by this retaining enzyme were measured, absolute parameters being obtained by active-site titration with 2´,4´,6´-trinitrophenyl 2-deoxy-2,2-difluoro-α-d-galactopyranoside. The variation of kcat/Km for 1-naphthyl-α-d-galactopyranoside with pH is bell-shaped, with pK1 = 1.91 and pK2 = 5.54. The αD(V/K) value for p-nitrophenyl-α-d-glucopyranoside is 1.031±0.007 at the optimal pH of 3.75 and 1.114±0.006 at pH 7.00, indicating masking of the intrinsic effect at optimal pH. There is no α-2H effect on binding galactose [αD(Ki) = 0.994±0.013]. The enzyme hydrolyses p-nitrophenyl β-l-arabinopyranoside ∼ 510 times slower than the galactoside, but has no detectable activity on the α-d-glucopyranoside or α-d-mannopyranoside. Hydrolysis of α-galactosides with poor leaving groups is Michaelian, but that of substrates with good leaving groups exhibits pronounced apparent substrate inhibition, with Kis values similar to Km values. We attribute this to the binding of the second substrate molecule to a β-galactopyranosyl-enzyme intermediate, forming an E•βGal•αGalX complex which turns over slowly, if at all. 1-Fluoro-α-d-galactopyranosyl fluoride, unlike α-d-galactopyranosyl fluoride, is a Michaelian substrate, indicating that the effect of 1-fluorine substitution is greater on the first than on the second step of the enzyme reaction.
Substrate inhibition and pH effect on denitritation with granular biomass
