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

Maria Hrmova,E Ann Macgregor,Peter Biely,Richard J Stewart,Geoffrey B Fincher

doi:10.1074/jbc.273.18.11134

Maria Hrmova, E Ann Macgregor + Show 3 more

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https://doi.org/10.1074/jbc.273.18.11134

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Abstract

A beta-glucosidase, designated isoenzyme betaII, from germinated barley (Hordeum vulgare L.) hydrolyzes aryl-beta-glucosides and shares a high level of amino acid sequence similarity with beta-glucosidases of diverse origin. It releases glucose from the non-reducing termini of cellodextrins with catalytic efficiency factors, kcat/Km, 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 beta-glucosidases (EC 3.2.1.21) and the polysaccharide exohydrolase, (1,4)-beta-glucan glucohydrolase (EC 3.2.1.74). At high concentrations (100 mM) of 4-nitrophenyl beta-glucoside, beta-glucosidase isoenzyme betaII catalyzes glycosyl transfer reactions, which generate 4-nitrophenyl-beta-laminaribioside, -cellobioside, and -gentiobioside. Subsite mapping with cellooligosaccharides indicates that the barley beta-glucosidase isoenzyme betaII has six substrate-binding subsites, each of which binds an individual beta-glucosyl residue. Amino acid residues Glu181 and Glu391 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 (beta/alpha)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.

Highlights

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
The Brownlee C18 guard column was obtained from Applied Biosystems (Foster City, CA), a W-Porex C18 analytical column was from Phenomenex (Torrance, CA), Kieselgel 60 thin layer plates and sodium 2,2-dimethyl-2-silapentane-5-sulfonic acid were from Merck (Darmstadt, Germany), chromatography paper no. 3MM Chr was from Whatman (Maidstone, Kent, United Kingdom), and (1,4)-␤oligoglucosides of degree of polymerization (DP) 2– 6 and (1,3)-␤-oligoglucosides of DP 2–7 were from Seikagaku Kogyo Co. (Tokyo, Japan)
Kinetic analyses of ␤-glucosidase isoenzyme ␤II from barley have shown that the enzyme rapidly hydrolyzes (1,4)-␤-oligoglucosides and that the catalytic efficiency factor, kcat/Km, increases as the DP of the substrate increases (Table I)

Summary

Introduction

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, kcat/Km, that increase approximately 9-fold as the degree of polymerization of these substrates increases from 2 to 6. The increased hydrolytic rate with oligosaccharides is a characteristic often observed with polysaccharide exohydrolases (5), the barley ␤-glucosidase is not able to hydrolyze polymeric (1,4)-␤-glucans (2, 3) These apparent anomalies led Hrmova et al (3) to suggest that the barley enzyme could be classified either as a polysaccharide exohydrolase of the (1,4)-␤-glucan glucohydrolase group (EC 3.2.1.74) or as a ␤-glucosidase of the EC 3.2.1.21 class. The substrate specificity, action pattern, putative catalytic residues, and subsite mapping data can all be reconciled with a three-dimensional model of the barley ␤-glucosidase, which is based on the x-ray crystal structure of an homologous cyanogenic ␤-glucosidase from white clover (6)

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