Substrate Binding and Catalytic Mechanism of a Barley β-d-Glucosidase/(1,4)-β-d-Glucan Exohydrolase

Abstract

A ␤-glucosidase, designated isoenzyme ␤II, from germinated barley (Hordeum vulgare L.) hydrolyzes aryl-␤-glucosides and shares a high level of amino acid sequence similarity with ␤-glucosidases of diverse origin. It releases glucose from the non-reducing termini of cellodextrins with catalytic efficiency factors, k cat /K m , that increase approximately 9-fold as the degree of polymerization of these substrates increases from 2 to 6. Thus, the enzyme has a specificity and action pattern characteristic of both ␤-glucosidases (EC 3.2.1.21) and the polysaccharide exohydrolase, (1,4)-␤-glucan glucohydrolase (EC 3.2.1.74). At high concentrations (100 mM) of 4-nitrophenyl ␤-glucoside, ␤-glucosidase isoenzyme ␤II catalyzes glycosyl transfer reactions, which generate 4-nitrophenyl-␤-laminaribioside, -cellobioside, and -gentiobioside. Subsite mapping with cellooligosaccharides indicates that the barley ␤-glucosidase isoenzyme ␤II has six substrate-binding subsites, each of which binds an individual ␤-glucosyl residue. Amino acid residues Glu 181 and Glu 391 are identified as the probable catalytic acid and catalytic nucleophile, respectively. The enzyme is a family 1 glycoside hydrolase that is likely to adopt a (␤/␣) 8 barrel fold and in which the catalytic amino acid residues appear to be located at the bottom of a funnel-shaped pocket in the enzyme.Two ␤-glucosidases of apparent molecular mass 62,000 have been purified from extracts of germinated barley grain (1-3) and can be classified in the family 1 group of glycosyl hydrolases and related enzymes (4). The two enzymes have been designated isoenzymes ␤I and ␤II, and have isoelectric points of 8.9 and 9.0, respectively. Amino acid sequence analyses reveal a single amino acid difference in the first 50 NH 2 -terminal amino acid residues, and the complete amino acid sequence of isoenzyme ␤II has been deduced from the nucleotide sequence of a corresponding cDNA clone (2).The function of the ␤-glucosidases in the germinated barley grain has not been defined unequivocally, but will clearly be related to their substrate specificities. It has been widely assumed that ␤-glucosidases prefer substrates of the type G-O-X, where G indicates the glucosyl residue and X can either be another glycosyl residue, for which the linkage position is not crucial, or a non-glycosyl aglycone group. As a result of the capacity of many ␤-glucosidases to hydrolyze glucosides with a range of glycosyl or non-glycosyl aglycone groups, non-physiological substrates such as 4-nitrophenyl ␤-D-glucopyranoside (4-NPG) 1 have been synthesized to measure activity in convenient spectrophotometric assays; the barley enzymes have also been assayed in this way. It has further been assumed that the rate of hydrolysis of oligomeric substrates by ␤-glucosidases will remain approximately constant or decrease with increasing degree of polymerization (DP) of the substrate (5). However, the barley ␤-glucosidase isoenzyme ␤II hydrolyzes (1,4)-␤-oligoglucosides much more efficiently than it hydrolyzes the aryl-␤-gluco...

Structural Basis for Broad Substrate Specificity in Higher Plant β-d-Glucan Glucohydrolases

“…The major product was 4NP-␤-gentiobioside, and the trisaccharide 4NP-␤-gentiotrioside also was detected. The structures of these oligoglucosides were confirmed (data not shown) by electrospray ionization mass spectrometry and 13 C-NMR spectroscopy (Hrmova et al, 1998b). …”

“…The compounds were excised from the 1048 The Plant Cell chromatogram, eluted with water, concentrated under reduced pressure, and subjected to analyses by electrospray ionization mass spectrometry and 13 C-NMR spectroscopy, as described previously (Hrmova et al, 1998b).…”

“…to define the number of subsites and to calculate the binding affinities of individual ␤ -D -glucosyl binding subsites in the barley ␤ -D -glucan glucohydrolase (Hrmova et al, 1995(Hrmova et al, , 1998b.…”

“…Alternatively, in the third step, the covalent glycosyl-enzyme intermediate (E . Glc) can be cleaved by an activated substrate molecule (R-OH), leading to a glycosyl transfer product (E ‫ف‬ Glc-OR), which remains noncovalently bound to the enzyme and is released (k 4 ) when a second substrate molecule approaches the active site and forms the next Michaelis complex (E -S).Broad-Specificity ␤ -D -Glucan Glucohydrolases 1035 to define the number of subsites and to calculate the binding affinities of individual ␤ -D -glucosyl binding subsites in the barley ␤ -D -glucan glucohydrolase (Hrmova et al, 1995(Hrmova et al, , 1998b. …”

“…Subsites are labeled from Ϫ1 to ϩ2, where the Ϫ1 subsite binds a nonreducing glucosyl residue of the substrate. Subsite affinities (A i ) were calculated using k cat ·K m Ϫ1 values (Table 3) as described previously (Hrmova et al, 1998b). …”