Subsite structure of exo-1,4-β-glucosidase from Acetobacter xylinum BPR2001

Abstract

The Michaelis constant ( K m) and molecular activity ( k 0) of an exo-1,4-β-glucosidase (EC 3.2.1.74) from Acetobacter xylinum subsp. sucrofermentans BPR2001 for hydrolysis of cello-oligosaccharides (G2–G6) were determined by steady-state kinetic analysis. The 1 K m and k 0 values for G2 were much lower than those for G3–G6. The enzyme was competitively inhibited by glucono-δ-lactone and conduritol-β epoxide. Based on the theory of Hiromi et al. (Biochim. Biophys. Acta, 302: 362–375, 1973), the subsite affinities (A i, i=1–6) and the intrinsic hydrolysis rate constant for substrate linkage in a productive complex ( k int) of the enzyme were kinetically estimated: A 1=2.46 kcal/mol, A 2=−0.44 kcal/mol, A 3=3.70 kcal/mol, A 4=0.33 kcal/mol, A 5=0.27 kcal/mol, A 6=0.06 kcal/mol, and k int=33.4 s −1. The subsite affinities were different from those for the β-glucosidase from Aspergillus niger and for the exo-1,4-β-glucosidase from Torulopsis wickerhamii, in that the A 2 value for the Acetobacter enzyme was negative. These results suggest that the enzyme possesses subsite affinities which have never previously been reported among exo-type glucosidases.