Investigation of the active site of the cyanogenic beta-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 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 (ki/Ki = 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 pKa 7.0, which is very similar to that of the acid catalyst group of the enzyme (pKE2 = 7.2). The involved amino acid, which has to be ionized for the inactivation, was identified as Glu-198 using 14C-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 A1-A5 cellulases and lies in a consensus Asn-Glu-Pro motif occurring in all of these enzymes.
Keresztessy
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Department of Biochemistry and Genetics, The University, Newcastle upon Tyne, United Kingdom.
Kiss
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Hughes
M A
MA
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D005971:Glutamates / Q000737:chemistry; D007700:Kinetics; D002365:Manihot / Q000201:enzymology*; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D000345:Affinity Labels; D005971:Glutamates; C090780:N-bromoacetylglucopyranosylamine; C021514:cyanogenic beta-glucosidase; D001617:beta-Glucosidase; D000117:Acetylglucosamine