INVESTIGATION OF THE ACTIVE-SITE OF THE CYANOGENIC BETA-D-GLUCOSIDASE(LINAMARASE) FROM MANIHOT-ESCULENTA CRANTZ (CASSAVA) .2. IDENTIFICATION OF GLU-198 AS AN ACTIVE-SITE CARBOXYLATE GROUP WITH ACID CATALYTIC FUNCTION

The broad-specificity cyanogenic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) from Manihot esculenta Crant z (cassava) was irreversibly inactivated by N-bromoacetyl-beta-D-gluco pyranosylamine 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-beta-D-glucopyranosidase, p-nitrophenyl-beta-D-galactop yranosidase, and linamarase activities of the enzyme. The competitive inhibitor p-nitrothiophenyl-beta-D-glucopyranoside protected the enzym e from inactivation. pH dependence of the pseudo-first-order rate cons tant of inactivation revealed the involvement of an amino acid side ch ain in the inactivation process with pK(a) 7.0, which is very similar to that of the acid catalyst group of the enzyme (pK(2)(E) = 7.2). The involved amino acid, which has to be ionized for the inactivation, wa s identified as Glu-198 using C-14-labeled inactivator to label the en zyme, cleaving the labeled protein into peptides and then purifying an d sequencing the labeled peptide. This residue is highly conserved in the homologous family A beta-glucosidases and family A(1)-A(5) cellula ses and lies in a consensus Asn-Glu-Pro motif occurring in all of thes e enzymes. (C) 1994 Academic Press, Inc.