Investigation of the Active Site of the Cyanogenic β-D-Glucosidase (Linamarase) from Manihot esculenta Crantz (Cassava). II. Identification of Glu-198 as an Active Site Carboxylate Group with Acid Catalytic Function

The broad-specificity cyanogenic β-D-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) from Manihot esculenta Crantz (cassava) was irreversibly inactivated by N -bromoacetyl-β-D-glucopyranosylamine according to pseudo-first-order kinetics with a second-order efficiency constant ( k i / K i = 0.1 min −1 M −1 ) identical for p -nitrophenyl-β-D-glucopyranosidase, p -nitrophenyl-β-D-galactopyranosidase, and linamarase activities of the enzyme. The competitive inhibitor p -nitrothiophenyl-β-D-glucopyranoside protected the enzyme from inactivation. pH dependence of the pseudo-first-order rate constant of inactivation revealed the involvement of an amino acid side chain in the inactivation process with pK a 7.0, which is very similar to that of the acid catalyst group of the enzyme ( pK E 2 = 7.2). The involved amino acid, which has to be ionized for the inactivation, was identified as Glu-198 using 14 C-labeled inactivator to label the enzyme, cleaving the labeled protein into peptides and then purifying and sequencing the labeled peptide. This residue is highly conserved in the homologous family A β-glucosidases and family A 1 -A 5 cellulases and lies in a consensus Asn-Glu-Pro motif occurring in all of these enzymes.