Identification of a cold-adapted and metal-stimulated ß-1,4-glucanase with potential use in the extraction of bioactive compounds from plants.

Cold-adapted endo-ß-1,4-glucanases hold great potential for industrial processes requiring high activity at mild temperatures such as in food processing and extraction of bioactive compounds from plants. Here, we identified and explored the specificity, mode of action, kinetic behavior, molecular structure and biotechnological application of a novel endo-ß-1,4-glucanase (XacCel8) from the phytopathogen Xanthomonas citri subsp. citri. This enzyme belongs to an uncharacterized phylogenetic branch of the glycoside hydrolase family 8 (GH8) and specifically cleaves internal ß-1,4-linkages of cellulose and mixed-linkage ß-glucans releasing short cello-oligosaccharides ranging from cellobiose to cellohexaose. XacCel8 acts in near-neutral pHs and in a broad temperature range (10-50 °C), which are distinguishing features from conventional thermophilic ß-1,4-glucanases. Interestingly, XacCel8 was greatly stimulated by cobalt ions, which conferred higher conformational stability and boosted the enzyme turnover number. The potential application of XacCel8 was demonstrated in the caffeine extraction from guarana seeds, which improved the yield by 2.5 g/kg compared to the traditional hydroethanolic method (HEM), indicating to be an effective additive in this industrial process. Therefore, XacCel8 is a metal-stimulated and cold-adapted endo-ß-1,4-glucanase that could be applied in a diverse range of biotechnological processes under mild conditions such as caffeine extraction from guarana seeds.

Enzymatic Characteristics of a Highly Thermostable ß-(1-4)-Glucanase from Fervidobacterium islandicum AW-1 (KCTC 4680).

A highly thermostable ß-(1-4)-glucanase (NA23_08975) gene (fig) from Fervidobacterium islandicum AW-1, a native-feather degrading thermophilic eubacterium, was cloned and expressed in Escherichia coli. The recombinant FiG (rFiG) protein showed strong activity toward ß-D-glucan from barley (367.0 IU/mg), galactomannan (174.0 IU/mg), and 4-nitrophenyl-cellobioside (66.1 IU/mg), but relatively weak activity was observed with hydroxyethyl cellulose (5.3 IU/mg), carboxymethyl cellulose (2.4 IU/mg), and xylan from oat spelt (1.4 IU/mg). rFiG exhibited optimal activity at 90°C and pH 5.0. In addition, this enzyme was extremely thermostable, showing a half-life of 113 h at 85°C. These results indicate that rFiG could be used for hydrolysis of cellulosic and hemicellulosic biomass substrates for biofuel production.

The family 22 carbohydrate-binding module of bifunctional xylanase/ß-glucanase Xyn10E from Paenibacillus curdlanolyticus B-6 has an important role in lignocellulose degradation.

A newly isolated endo-ß-1,4-xylanase (Xyn10E) from Paenibacillus curdlanolyticus B-6 has a modular structure consisting of a family 22 carbohydrate-binding module (CBM), a glycoside hydrolase (GH) family 10 catalytic domain, two fibronectin type III (Fn3) domains, and a family 3 CBM at the C-terminus. Intact Xyn10E (rXyn10E), CBM22-deleted Xyn10E (X-CBM3), CBM3-deleted Xyn10E (X-CBM22), and GH10 catalytic domain only (X-GH10) were expressed in Escherichia coli. rXyn10E showed bifunctional degradation activity toward xylan and ß-glucan and also degraded microcrystalline cellulose. Although X-CBM3 and X-GH10 had drastically reduced xylanase and ß-glucanase activities, X-CBM22 mostly retained these activities. Similar Km values were obtained for rXyn10E and X-CBM3, but kcat and kcat/Km values for X-CBM3 and X-GH10 were lower than those for rXyn10E, suggesting that CBM22 of Xyn10E may contribute to catalytic efficiency. In binding assays, X-CBM3 was still able to bind to ß-glucan, soluble xylan, insoluble xylan, and cellulose through GH10 and CBM3. These results indicate that CBM22 has an important role not only in binding to xylan and ß-glucan but also in feeding both polysaccharides into the neighboring GH10 catalytic domain. rXyn10E showed remarkable synergism with rXyn11A, a major xylanase subunit of P. curdlanolyticus B-6, in the degradation of untreated corn stover and sugarcane bagasse; however, the combination of X-CBM3 and rXyn11A was not synergistic. These results indicate that Xyn10E and Xyn11A act synergistically on lignocellulosic biomass, and CBM22 is essential for efficient degradation of lignocellulosic materials.

Biochemical characterization, molecular cloning, and structural modeling of an interesting ß-1,4-glucanase from Sclerotinia sclerotiorum.

The filamentous fungus Sclerotinia sclerotiorum produces a complete set of cellulolytic enzymes needed for efficient solubilization of native cellulose, the major component of plants. In this work, we reported the molecular characterization of an important glycosyl-hydrolase enzyme classified as endo-ß-1,4-glucanase. The importance of this enzyme was revealed with the in-gel activity staining, showing a high degradation capacity of cellulose. When purified from native gel and ran in denaturing polyacrylamide gel, the polypeptide has an apparent molecular mass of about 34 kDa called Endo2. For further characterization of this protein, a mass spectrometry approach was carried out. The LC-MS/MS analysis revealed two peptides belonging to this enzyme. The genomic DNA and cDNA sequences were resolved by PCR amplification and sequencing, revealing a gene with two intron sequences. The open reading frame of 987 bp encoded a putative polypeptide of 328 amino acids having a calculated molecular mass of 33,297 Da. Yet, the molecular modeling and comparative investigation of different 3D cellulase structures showed that this endoglucanase isoform has probably two domains. A core domain having a high similarity with endoglucanases family 5 and a cellulose-binding domain having similarities with those of exo-type cellulases of family 1, linked together by a serine-threonine-rich region. These results are with great interests and show new characteristics of S. sclerotiorum glucanase.

Cloning, characterization and molecular docking of a highly thermostable ß-1,4-glucosidase from Thermotoga petrophila.

A genomic DNA fragment, encoding a thermotolerant ß-glucosidase, of the obligate anaerobe Thermotoga petrophila RKU-1 was cloned after PCR amplification into Escherichia coli strain BL21 CodonPlus. The purified cloned enzyme was a monomeric, 51.5 kDa protein (by SDS-PAGE) encoded by 1.341 kb gene. The estimated K (m) and V (max) values against p-nitrophenyl-ß-D-glucopyranoside were 2.8 mM and 42.7 mmol min(-1) mg(-1), respectively. The enzyme was also active against other p-nitrophenyl substrates. Possible catalytic sites involved in hydrolyzing different p-nitrophenyl substrates are proposed based on docking studies of enzyme with its substrates. Because of its unique characters, this enzyme is a potential candidate for industrial applications.

Structure and activity of exo-1,3/1,4-ß-glucanase from marine bacterium Pseudoalteromonas sp. BB1 showing a novel C-terminal domain.

UNLABELLED: Following the discovery of an exo-1,3/1,4-ß-glucanase (glycoside hydrolase family 3) from a seaweed-associated bacterium Pseudoalteromonas sp. BB1, the recombinant three-domain protein (ExoP) was crystallized and its structure solved to 2.3 Å resolution. The first two domains of ExoP, both of which contribute to the architecture of the active site, are similar to those of the two-domain barley homologue ß-d-glucan exohydrolase (ExoI) with a distinctive Trp-Trp clamp at the +1 subsite, although ExoI displays broader specificity towards ß-glycosidic linkages. Notably, excision of the third domain of ExoP results in an inactive enzyme. Domain 3 has a ß-sandwich structure and was shown by CD to be more temperature stable than the native enzyme. It makes relatively few contacts to domain 1 and none at all to domain 2. Two of the domain 3 residues involved at the interface, Q683 (forming one hydrogen bond) and Q676 (forming two hydrogen bonds) were mutated to alanine. Variant Q676A retained about half the activity of native ExoP, but the Q683A variant was severely attenuated. The crystal structure of Q683A-ExoP indicated that domain 3 was highly mobile and that Q683 is critical to the stabilization of ExoP by domain 3. Small-angle X-ray scattering data lent support to this proposal. Domain 3 does not appear to be an obvious carbohydrate-binding domain and is related neither in sequence nor structure to the additional domains characterized in other glycoside hydrolase 3 subgroups. Its major role appears to be for protein stability but it may also help orient substrate. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 3UT0, 3USZ, 3F95 and 3RRX.

Cloning and characterization of a novel cold-active endoglucanase establishing a new subfamily of glycosyl hydrolase family 5 from a psychrophilic deep-sea bacterium.

The gene of a novel endo-ß-1,4-glucanase (named Cel5M) was isolated from the psychrophilic deep-sea bacteria Pseudomonas sp. MM15. The deduced protein sequence lacked the typical cellulase domain structures of the carbohydrate-binding module and the linker region. Cel5M showed relatively higher activity toward carboxymethyl cellulose, but much lower activity toward p-nitrophenyl-ß-D-galactopyranoside and no activity toward avicel. Cel5M was identified as a cold-active cellulase with an optimal temperature of 30 °C and it was active within a narrow pH range with an optimum at pH 4.5. Phylogenetic analysis showed that Cel5M represented a new subfamily of the glycosyl hydrolase family 5, representing an opportunity for research into and applications of novel cold-active cellulases.

Thermal denaturation of ß-glucosidase B from Paenibacillus polymyxa proceeds through a Lumry-Eyring mechanism.

ß-glucosidase B (BglB), 1,4-ß-D: -glucanohydrolase, is an enzyme with various technological applications for which some thermostable mutants have been obtained. Because BglB denatures irreversibly with heating, the stabilities of these mutants are assessed kinetically. It, therefore, becomes relevant to determine whether the measured rate constants reflect one or several elementary kinetic steps. We have analyzed the kinetics of heat denaturation of BglB from Paenibacillus polymyxa under various conditions by following the loss of secondary structure and enzymatic activity. The denaturation is accompanied by aggregation and an initial reversible step at low temperatures. At T ≥ T ( m ), the process follows a two-state irreversible mechanism for which the kinetics does not depend on the enzyme concentration. This behavior can be explained by a Lumry-Eyring model in which the difference between the rates of the irreversible and the renaturation steps increases with temperature. Accordingly, at high scan rates (≥1 °C min(-1)) or temperatures (T ≥ T ( m )), the measurable activation energy involves only the elementary step of denaturation.

Fluorescence study on interactions of alpha-crystallin with the molten globule state of 1, 4-beta-D-glucan glucanohydrolase from Thermomonospora sp. induced by guanidine hydrochloride.

In this paper, the interaction between alpha- crystallin and molten globule structure of 1,4-beta-D-Glucan Glucohydrolase (TSC) from an alkalothermophilic Thermomonospora sp. was investigated mainly by fluorescence quenching spectra, circular dichroism and three dimensional fluorescence spectra under simulative physiological conditions. Denaturation studies using GdnCl indicated that TSC folds through a partially folded state that resembles molten globule at 1.8 M GdnCl. The chaperone activity of alpha- crystallin was employed to study refolding of TSC. Here we studied the refolding of GdnCl denatured TSC from its molten globule state (TSC-m complex) in the presence and absence of alpha-crystallin to elucidate the molecular mechanism of chaperone-mediated in vitro folding. Our results, based on intrinsic tryptophan fluorescence and ANS binding studies, suggest that alpha-crystallin formed a complex with a putative intermediate molten globule-like intermediate in the refolding pathway of TSC. Reconstitution of the active TSC was observed on cooling the alpha-crystallin * TSC-m complex to 4 degrees C. Addition of alpha-crystallin to the molten globule-like intermediate of TSC (TSC-m complex) complex initiated the refolding of TSC with 69% recovery of the biological activity of the enzyme.

Improved catalytic efficiency of endo-beta-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution.

Bacillus subtilis endo-beta-1,4-glucanase (Cel5A) hydrolyzes cellulose by cleavage of the internal bonds in the glucose chains, producing new ends randomly. Using directed evolution techniques of error-prone polymerase chain reaction (PCR) and DNA shuffling, several Cel5A variants with improved catalytic activity had been screened from the mutant library, which contained 71,000 colonies. Compared with the wild-type enzyme, the variants (M44-11, S75 and S78) showed 2.03 to 2.68-fold increased activities toward sodium carboxymethyl cellulose (CMC), while the M44-11 also exhibited a wider pH tolerance and higher thermostability. Structural models of M44-11, S75, S78, and WT proteins revealed that most of the substitutions were not located in the strictly conserved regions, except the mutation V255A of S75, which was closed to the nucleophile Glu257 in the catalytic center of the enzyme. Moreover, V74A and D272G of M44-11, which were not located in the substrate binding sites and the catalytic center, might result in improved stability and catalytic activity. These results provided useful references for directed evolution of the enzymes that belonged to the glycoside hydrolase family 5 (GH5).

Characterization and action patterns of two beta-1,4-glucanases purified from cellulomonas uda CS1-1.

Two beta-1,4-glucanases (DI and DIII fractions) were purified to homogeneity from the culture filtrate of a cellulolytic bacteria, Cellulomonas sp. CS1-1, which was classified as a novel species belonging to Cellulomonas uda based on chemotaxanomic and phylogenetic analyses. The molecular mass was estimated as 50,000 Da and 52,000 Da for DI and DIII, respectively. Moreover, DIII was identified as a glycoprotein with a pI of 3.8, and DI was identified as a non-glycoprotein with a pI of 5.3. When comparing the ratio of the CMC-saccharifying activity and CMC-liquefying activity, DI exhibited a steep slope, characteristic of an endoglucanase, whereas DIII exhibited a low slope, characteristic of an exoglucanase. The substrate specificity of the purified enzymes revealed that DI efficiently hydrolyzed CMC as well as xylan, whereas DIII exhibited a high activity on microcrystalline celluloses, such as Sigmacells. A comparison of the hydrolysis patterns for pNP-glucosides (DP 2-5) using an HPLC analysis demonstrated that the halosidic bond 3 from the nonreducing end was the preferential cleavage site for DI, whereas bond 2, from which the cellobiose unit is split off, was the preferential cleavage site for DIII. The partial N-terminal amino acid sequences for the purified enzymes were 1Ala-Gly-Ser-Thr-Leu-Gln-Ala-Ala-Ala-Ser-Glu-Ser-Gly-Arg-Tyr15- for DI and 1Ala-Asp-Ser-Asp-Phe-Asn-Leu-Tyr-Val-Ala-Glu-Asn-Ala-Met-Lys15- for DIII. The apparent sequences exhibited high sequence similarities with other bacterial beta-1,4-glucanases as well as beta-1,4-xylanases.

Biochemical characterization of a novel beta-1--3, 1--4 glucan 4-glucanohydrolase from Thermomonospora sp. having a single active site for lichenan and xylan.

A bifunctional high molecular weight (Mr, 64,500 Da) beta-1-3, 1-4 glucan 4-glucanohydrolase was purified to homogeneity from Thermomonospora sp., exhibiting activity towards lichenan and xylan. A kinetic method was used to analyze the active site that hydrolyzes lichenan and xylan. The experimental data was in agreement with the theoretical values calculated for a single active site. Probing the conformation and microenvironment at active site of the enzyme by fluorescent chemo-affinity label, OPTA resulted in the formation of an isoindole derivative with complete inactivation of the enzyme to hydrolyse both lichenan and xylan confirmed the results of kinetic method. OPTA forms an isoindole derivative by cross-linking the proximal thiol and amino groups. The modification of cysteine and lysine residues by DTNB and TNBS respectively abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating the participation of cysteine and lysine in the formation of isoindole complex.

Degradation of cellulosome-produced cello-oligosaccharides by an extracellular non-cellulosomal beta-glucan glucohydrolase, BglA, from Clostridium cellulovorans.

Clostridium cellulovorans degrades cellulose efficiently to small oligosaccharides, which are used as an energy source. To characterize enzymes related to degrading small oligosaccharides, a gene was cloned for an extracellular non-cellulosomal beta-glucan glucohydrolase (BglA) classified as a family-1 glycosyl hydrolase in C. cellulovorans. Recombinant BglA (rBglA) had higher activity on long glucooligomers than on cellobiose. When cellulosomes and rBglA were incubated with cellulose, the oligosaccharides produced were degraded more effectively to cellobiose and glucose, than with cellulosomes alone, indicating that BglA facilitated the degradation of accessible cello-oligosaccharides produced from cellulose by C. cellulovorans cellulosomes. Thus, this is an example of an extracellular non-cellulosomal enzyme working in a cooperative manner with cellulosomes to degrade cellulose to sugars.

Conformation and microenvironment of the active site of a low molecular weight 1,4-beta-D-glucan glucanohydrolase from an alkalothermophilic Thermomonospora sp.: involvement of lysine and cysteine residues.

Conformation and microenvironment at the active site of 1,4-beta-D-glucan glucanohydrolase was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde. OPTA has been known to form a fluorescent isoindole derivative by cross-linking the proximal thiol and amino groups of cysteine and lysine. Modification of lysine of the enzyme by TNBS and of cysteine residue by PHMB abolished the ability of the enzyme to form an isoindole derivative with OPTA. Kinetic analysis of the TNBS and PHMB-modified enzyme suggested the presence of essential lysine and cysteine residues, respectively, at the active site of the enzyme. The substrate protection of the enzyme with carboxymethylcellulose (CMC) confirmed the involvement of lysine and cysteine residues in the active site of the enzyme. Multiple sequence alignment of peptides obtained by tryptic digestion of the enzyme showed cysteine is one of the conserved amino acids corroborating the chemical modification studies.

Crystallization and preliminary X-ray characterization of a thermostable low-molecular-weight 1,4-beta-D-glucan glucohydrolase from an alkalothermophilic Thermomonospora sp.

Cellulases catalyze the hydrolysis of beta-1,4-glycosidic linkages within cellulose, the most abundant organic polymer on earth. The cellulase (TSC; EC 3.2.1.4) from an alkalothermophilic Thermomonospora sp. has a low molecular weight of 14.2 kDa. It is optimally active at 323 K and stable over the wide pH range of 5-9. Moreover, it has bifunctional activity against cellulose and xylan polymers. In this study, TSC was purified from the native source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.9, b = 79.5, c = 99.7 angstroms, and diffract to better than 2.3 angstroms resolution.

Reconstitution of cyanogenesis in barley (Hordeum vulgare L.) and its implications for resistance against the barley powdery mildew fungus.

Barley (Hordeum vulgare L.) produces a leucine-derived cyanogenic beta-D-glucoside, epiheterodendrin that accumulates specifically in leaf epidermis. Barley leaves are not cyanogenic, i.e. they do not possess the ability to release hydrogen cyanide, because they lack a cyanide releasing beta-D-glucosidase. Cyanogenesis was reconstituted in barley leaf epidermal cells through single cell expression of a cDNA encoding dhurrinase-2, a cyanogenic beta-D-glucosidase from sorghum. This resulted in a 35-60% reduction in colonization rate by an obligate parasite Blumeria graminis f. sp. hordei, the causal agent of barley powdery mildew. A database search for barley homologues of dhurrinase-2 identified a (1,4)-beta-D-glucan exohydrolase isozyme betaII that is located in the starchy endosperm of barley grain. The purified barley (1,4)-beta-D-glucan exohydrolase isozyme betaII was found to hydrolyze the cyanogenic beta-D-glucosides, epiheterodendrin and dhurrin. Molecular modelling of its active site based on the crystal structure of linamarase from white clover, demonstrated that the disposition of the catalytic active amino acid residues was structurally conserved. Epiheterodendrin stimulated appressoria and appressorial hook formation of B. graminis in vitro, suggesting that loss of cyanogenesis in barley leaves has enabled the fungus to utilize the presence of epiheterodendrin to facilitate host recognition and to establish infection.

Purification and properties of a low molecular weight 1,4-beta-d-glucan glucohydrolase having one active site for carboxymethyl cellulose and xylan from an alkalothermophilic Thermomonospora sp.

A low molecular weight 1,4-beta-D-glucan glucohydrolase from an extracellular culture filtrate of Thermomonospora sp. was purified to homogeneity. The molecular weight of the purified enzyme was 14.2 kDa by MALDI-TOF analysis and is in agreement with SDS-PAGE and gel filtration chromatography. The purified enzyme exhibited both endocarboxymethyl cellulase and endoxylanase activities. A kinetic method was employed to study the active site of the enzyme that hydrolyzes both carboxymethyl cellulose and xylan. The experimental data coincide well with the theoretical values calculated for the case of a single active site. Conformation and microenvironment at the active site was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde as the chemical initiator. Formation of isoindole derivative resulted in complete inactivation of the enzyme to hydrolyze both xylan and CMC as judged by fluorescence studies corroborating a single active site for the hydrolysis of xylan and CMC.

Improved catalytic efficiency and active site modification of 1,4-beta-D-glucan glucohydrolase A from Thermotoga neapolitana by directed evolution.

Thermotoga neapolitana 1,4-beta-d-glucan glucohydrolase A preferentially hydrolyzes cello-oligomers, such as cellotetraose, releasing single glucose moieties from the reducing end of the cello-oligosaccharide chain. Using directed evolution techniques of error-prone PCR and mutant library screening, a variant glucan glucohydrolase has been isolated that hydrolyzes the disaccharide, cellobiose, at a 31% greater rate than its wild type (WT) predecessor. The mutant library, expressed in Escherichia coli, was screened at 85 degrees C for increased hydrolysis of cellobiose, a native substrate rather than a chromogenic analog, using a continuous, thermostable coupled enzyme assay. The V(max) for the mutant was 108 +/- 3 units mg(-1), whereas that of the WT was 75 +/- 2 units mg(-1). The K(m) for both proteins was nearly the same. The k(cat) for the new enzyme increased by 31% and its catalytic efficiency (k(cat)/K(m)) for cellobiose also rose by 31% as compared with the parent. The nucleotide sequence of two positive clones and two null clones identified 11 single base shifts. The nucleotide transition in the most active clone caused an isoleucine to threonine amino acid substitution at position 170. Structural models for I170T and WT proteins were derived by sequence homology with Protein Data Bank code 1BGA from Paenibacillus polymyxa. Analysis of the WT and I170T model structures indicated that the substitution in the mutant enzyme repositioned the conserved catalytic residue Asn-163 and reconfigured entry to the active site.

A monovalent anion affected multi-functional cellulase EGX from the mollusca, Ampullaria crossean.

A cellulose hydrolytic enzyme was isolated from the stomach juice of Ampullaria crossean, a kind of herbivorous mollusca. The enzyme was purified 45.3-fold to homogenety by ammonium sulfate precipitation, DEAE-Sephadex A-50 column, Bio-gel P-100 gel filtration column, and phenyl-Sepharose CL-4B column chromatography. The enzyme was designated as cellulase EGX. The purified enzyme is a multi-functional enzyme with the activities of exo-beta-1,4-glucanase (14.84 U/mg for p-nitrophenyl beta-D-cellobioside), endo-beta-1,4-glucanase (40.3 U/mg for carboxymethyl cellulose), and endo-beta-1,4-xylanase (196 U/mg for soluble xylan from birchwood). The monovalent anions such as F(-), Cl(-), Br(-), I(-), and NO(3)(-) are essential for its exo-beta-1,4-glucanase activity but have no effect on the activity for xylan, while I(-) higher than 5mM would inhibit the exo-beta-1,4-glucanase activity. The monovalent anions Cl(-) and Br(-) activate its endo-beta-1,4-glucanase activity. Binding of Cl(-) enhances the thermostability of EGX, but does not affect its fluorescence emission spectrum. The molecular mass of EGX is 41.5 kDa, as determined by SDS-PAGE. The pI value is about pH 7.35. The xylan hydrolytic activity of EGX reaches to the maximum between pH 4.8 and 6.0 and the pNPC hydrolytic activity reaches the maximum between pH 4.8 and 5.6, while that for CMC hydrolytic activity is between pH 4.4 and 4.8. Preliminary results showed that the enzyme was secreted by the mollusca itself.

Can thioglycosides imitate the oxonium intermediate in glycosyl hydrolases?

Glycosyl hydrolases catalyse the acid hydrolysis of the glycosidic bond of glycans. The structure of barley beta-D-glucan glucohydrolase in complex with a thiol substrate analogue presents very short contacts between the carboxyl oxygen atoms of the catalytic acid and the sulphur atom of the inhibitor. The geometries of acetic acid and dimethylsulfide in various ionisation states from ab initio molecular orbital calculations predict similar short contacts when an acetate anion forms a complex with a sulphonium cation. The energy of this complex is, however, significantly greater than the energy of the complex where both acetic acid and dimethylsulfide are neutral. Calculations on an active site model of barley beta-D-glucan glucohydrolase indicate that the protein environment is able to significantly reduce this energy. The energy required for mechanical constraint of the short S...O separations, however, is identical to that required for the transfer of the proton from the acid to the sulphur. The identity of the species participating in the short contacts remains unanswered.