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

被引:39
作者
KERESZTESSY, Z
KISS, L
HUGHES, MA
机构
[1] UNIV NEWCASTLE UPON TYNE, DEPT BIOCHEM & GENET, NEWCASTLE UPON TYNE NE2 4HH, TYNE & WEAR, ENGLAND
[2] LAJOS KOSSUTH UNIV, INST BIOCHEM, H-4010 DEBRECEN, HUNGARY
关键词
BETA-GLUCOSIDASE; CASSAVA; IRREVERSIBLE INACTIVATION; AFFINITY LABELING; N-BROMOACETYL-BETA-D-GLUCOPYRANOSYLAMINE;
D O I
10.1006/abbi.1994.1507
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The broad-specificity cyanogenic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) from Manihot esculenta Crantz (cassava) was irreversibly inactivated by N-bromoacetyl-beta-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-beta-D-glucopyranosidase, p-nitrophenyl-beta-D-galactopyranosidase, and linamarase activities of the enzyme. The competitive inhibitor p-nitrothiophenyl-beta-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(2)(E) = 7.2). The involved amino acid, which has to be ionized for the inactivation, was identified as Glu-198 using C-14-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 beta-glucosidases and family A(1)-A(5) cellulases and lies in a consensus Asn-Glu-Pro motif occurring in all of these enzymes. (C) 1994 Academic Press, Inc.
引用
收藏
页码:323 / 330
页数:8
相关论文
共 54 条