Báo cáo khoa học: Substrate recognition by three family 13 yeast a-glucosidases Evaluation of deoxygenated and conformationally biased isomaltosides

Eur. J. Biochem. 269, 728–734 (2002) (cid:211) FEBS 2002

Substrate recognition by three family 13 yeast a-glucosidases Evaluation of deoxygenated and conformationally biased isomaltosides

Torben P. Frandsen1,*, Monica M. Palcic2 and Birte Svensson1

1Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark; 2Department of Chemistry, University of Alberta, Edmonton, Canada

found to be unimportant in this respect and very little or no contribution is indicated for all OH-groups of the reducing- end ring of the two a-glucosidases, probably reflecting their exposure to bulk solvent. The stereochemical course of hydrolysis by these three members of the retaining family 13 was confirmed by directly monitoring isomaltose hydrolysis using 1H NMR spectroscopy. Kinetic analysis of the hydrolysis of methyl 6-S-ethyl-a-isomaltoside and its 6-R- diastereoisomer indicates that a-glucosidase has 200-fold higher specificity for the S-isomer. Substrate molecular rec- ognition by these a-glucosidases are compared to earlier findings for the inverting, exo-acting glucoamylase from Aspergillus niger and a retaining a-glucosidase of glycoside hydrolase family 31, respectively.

Keywords: protein-carbohydrate interaction; NMR; glyco- sidase mechanism; substrate analogs; molecular recognition.

Important hydrogen bonding interactions between substrate OH-groups in yeast a-glucosidases and oligo-1,6-glucosidase from glycoside hydrolase family 13 have been identified by measuring the rates of hydrolysis of methyl a-isomaltoside and its seven monodeoxygenated analogs. The transition- state stabilization energy, DDG(cid:224), contributed by the indi- vidual OH-groups was calculated from the activities for the parent and the deoxy analogs, respectively, according to DDG(cid:224) (cid:136) –RT ln[(Vmax/Km)analog/(Vmax/Km)parent]. This analysis of the energetics gave DDG(cid:224) values for all three enzymes ranging from 16.1 to 24.0 kJÆmol)1 for OH-2¢, -3¢, -4¢, and -6¢, i.e. the OH-groups of the nonreducing sugar ring. These OH-groups interact with enzyme via charged hydro- gen bonds. In contrast, OH-2 and -3 of the reducing sugar contribute to transition-state stabilization, by 5.8 and 4.1 kJÆmol)1, respectively, suggesting that these groups participate in neutral hydrogen bonds. The OH-4 group is

these,

Strong intermolecular hydrogen bonds are very important in specificity of enzymes and other proteins that metabolize or bind carbohydrates [1–6]. Substrate analogs such as deoxygenated sugars, facilitate identification of critical contacts and enable quantification of the energetics of the protein–carbohydrate binding at the level of individual interacting sugar OH-groups and functional atoms or groups in the protein [4,7–11]. Alternatively, site-specific mutants of a protein are useful in evaluation of specific protein–carbohydrate interactions and further insight has been gained by combining mutant enzymes and analogs [7,9,10]. The binding energy contributed by substrate OH-groups has been determined for only a few carbohy- drate active enzymes. Of the starch hydrolase glucoamylase from Aspergillus niger has been the most intensively examined [7,9–13].

Three-dimensional structures of protein–carbohydrate complexes can guide and support protein engineering and molecular recognition experiments. For family 13 glycoside hydrolases, there are no crystal structures for a-glucosidases; however, the structure of free Bacillus oligo-1,6-glucosidase has been solved [14]. Furthermore, only a few a-glucosidases are produced by heterologous gene expression, which is a prerequisite for structure–function relationship investiga- tions by site-directed mutagenesis [15–21]. While the yeast genome is known and thus the primary structures of its a-glucosidases, the sequenced strain of Saccharomyces cerevisiae is not necessarily identical to the baker’s yeast used as a source of enzymes in the present study and sequences have not been reported for brewer’s yeast enzymes. In view of this limited information, use of synthetic substrate analogs is particularly attractive for gaining knowledge of the nature and strength of substrate– a-glucosidase interactions. Thus using deoxy-analogs key polar groups in maltose were identified for high pI barley a-glucosidase of glycoside hydrolase family 31 to be OH-4¢ and -6¢ with minor contributions for OH-3¢, -2¢, and -3 [13, 22].

Yeast a-glucosidase and oligo-1,6-glucosidase are exo- acting glycoside hydrolases catalyzing release of a-D-glucose from nonreducing ends of various a-linked substrates. The enzymes are further subclassified into type I, hydrolysing heterogeneous substrates like aryl glucosides and sucrose more efficiently than maltose; type II being highly active on maltose and isomaltose but of low activity toward aryl glucosides; and type III resembling type II, but hydrolysing

Correspondence to B. Svensson, Department of Chemistry, Carlsberg Laboratory, DK-2500 Copenhagen Valby, Denmark; Fax: + 45 33 27 47 08; Tel.: + 45 33 27 53 45; E-mail: bis@crc.dk Enzymes: a-glucosidase (a-D-glucoside glucohydrolase, EC 3.2.1.20); oligo-1,6-glucosidase (dextrin 6-a-glucanohydrolase, EC 3.2.1.10); glucoamylase (a-D-glucan glucohydrolase, EC 3.2.1.3). *Present address: Pantheco, Bøge Alle´ , DK 2970 Hørsholm Denmark. Dedication: this paper is dedicated to Prof. Joachim Thiem on the occasion of his 60th birthday. (Received 12 October 2001, revised 26 November 2001, accepted 30 November 2001)

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Substrate recognition in yeast a-glucosidases (Eur. J. Biochem. 269) 729

genated methyl a-isomaltosides [42], methyl 6-R-C-ethyl- and methyl 6-S-C-ethyl-a-isomaltoside [41] were generous gifts of U. Spohr and the late R. U. Lemieux, University of Alberta, Edmonton, Canada.

di- and oligo-saccharides and starch at comparable rates [23,24]. The sequence classifies a-glucosidases in glycoside hydrolase families 13 and 31 [25–27]. Yeast a-glucosidases and oligo-1,6-glucosidase belong to family 13 and are of type I that prefers p-nitrophenyl-a-D-glucopyranoside [28].

Enzyme assays

Glycoside hydrolase family 13 (or (cid:212)the a-amylase family(cid:213)) currently comprises 28 specificities of amylolytic and related enzymes. Several crystal structures of enzyme-inhibitor complexes highlight active sites created by b fi a segments in catalytic (b/a)8 barrel domains (reviewed in [29–31]). Because no ligand complex is available of oligo-1,6- glucosidase, the only structure-determined exo-acting a-glucosidase [14], side-chains participating in substrate binding and catalysis are solely identified by sequence comparison. Clearly a-glucosidases lack the sequence motif in b fi a loop 4 of family 13 [30] containing residues binding substrate at subsite +2 (nomenclature as in [32]) in a-amylases, cyclodextrin glycosyltransferases, and related enzymes [30,33–37].

In this study, seven monodeoxygenated isomaltosides are used to map substrate OH-groups required by yeast a-glucosidases and oligo-1,6-glucosidase in hydrolysis of the a-1,6-glucosidic bond. The energy contributed by each OH-group for transition-state stabilization reflects the strength of a specific protein–carbohydrate contact and energy profiles for the a-glucosidases and oligo-1,6-glucosi- dase are compared. 1H-NMR spectroscopy was used to confirm that all enzymes hydrolyse isomaltose with reten- tion of anomeric configuration characteristic of family 13 (reviewed in [38]).

a-Glucosidase activity was determined at 30 (cid:176)C in 0.1 M sodium maleate, pH 6.8 (oligo-1,6-glucosidase) or 50 mM phosphate, pH 6.8 (a-glucosidases). Glucose [7,10,40,41] was analysed for analogs at the reducing end ring (reaction volume 100 lL), aliquots (15 lL) being transferred at regular time intervals to microtiter plate wells already containing quench solution (200 lL 1 M Tris, pH 7.6, 5 UÆmL)1 glucose oxidase (A. niger), 1 UÆmL)1 peroxidase (horseradish), and 0.21 mgÆmL)1 o-dianisidine). Absor- bances were read at 450 nm after 1 h incubation at room temperature using a microtiter plate reader (Ceres UV900Hdi, Bio-Tek), and quantified using D-glucose as a standard [22,40]. Deoxygenated glucose analogs were ana- lysed essentially as described [10,40,41] with substrate analogs at the nonreducing end sugar (reaction volume 400 lL) aliquots (100 lL) were transferred to quench buffer containing 60 UÆmL)1 glucose oxidase, 1 UÆmL)1 peroxi- dase, and 0.1 mgÆmL)1 o-dianisidine, and the absorbances were read at 450 nm after 4 h incubation at room temperature, and quantified using the relevant deoxygenated D-glucose as standard. The a-glucosidase catalyzed hydro- lysis was initiated by addition of 0.1–91 U enzyme. The limited amounts of deoxygenated analogs available allowed only determination of second-order rate constants, Vmax/Km (s)1ÆU)1) (cid:136) vo/EoSo, where vo is the initial rate of hydro- lysis, So the initial substrate concentration, and Eo the amount of enzyme in U. Two So concentrations of around 0.1 · Km were used to ensure that substrate hydrolysis was linear with time. The increase in activation energy due to substrate deoxygenation was calculated by DDG(cid:224) (cid:136) –RTln[(Vmax/Km)a/(Vmax/Km)b] [43], where a refers to ana- log and b to parent substrate. For the two diastereoisomers, Vmax and Km were determined by fitting initial rates at eight different substrate concentrations from 0.1 · Km to 4 · Km to the Michealis–Menten equation essentially as described previously [40].

The exo-acting glucoamylase similarly to the a-glucosid- ases catalyses the releases of glucose from the nonreducing ends of substrates, but with inversion of the anomeric configuration [39]. While glucoamylase prefers the R-isomer of isomaltose diastereoisomeric analogs [40], a-glucosidase in the present study selects methyl 6-S-ethyl-a-isomaltoside in preference to the R-isomer. Molecular recognition of isomaltosides is more similar for the yeast a-glucosidase and oligo-1,6-glucosidase of glycoside of hydrolase family 13 when compared to that of glucoamylase of glycoside hydrolase family 15 or of a type II a-glucosidase from the retaining glycoside hydrolase family 31 [9,10,40,41].

M A T E R I A L S A N D M E T H O D S

Reaction stereochemistry

Enzymes and substrates

Lyophilized enzymes were redissolved in 0.1 M sodium phosphate pH 6.8 in D2O and the stereochemistry of isomaltose hydrolysis was determined by 1H NMR at 310 K using a Bruker AMX-600 spectrometer operated at 600 MHz. After recording the substrate spectrum of 100 mM isomaltose (in 600 lL 0.1 M phosphate, pH 6.8, in D2O), enzyme was added (oligo-1,6-glucosidase, 40 U; baker’s, 135 U and brewer’s yeast a-glucosidase, 140 U) and reactions monitored by recording spectra at regular intervals.

R E S U L T S A N D D I S C U S S I O N

Energetics of deoxy isomaltoside hydrolysis

(EC 3.2.1.10; Oligo-1,6-glucosidase from baker’s yeast Lot no. 23H8080), and a-glucosidases from brewer’s (EC 3.2.1.20; Type VI; Lot no. 21F8105) and baker’s (EC 3.2.1.20; Type I; Lot no. 122H8000) yeast were obtained from Sigma. After dissolution in 50 mM phosphate pH 6.8 (a-glucosidases) or 50 mM sodium maleate pH 6.8 (oligo-1,6-glucosidase) followed by extensive dialysis at 4 (cid:176)C against these buffers, the different enzymes (oligo-1,6- glucosidase, 30 UÆmL)1; brewer’s yeast a-glucosidase, 200 UÆmL)1; baker’s yeast a-glucosidase, 61 UÆmL)1) were used without further purification in the kinetic and stereo- chemical studies. One unit is defined as the amount of enzyme required to liberate 1 lmol of glucose from p-nitrophenyl a-D-glucoside (Sigma) per min at 30 (cid:176)C. The seven monodeoxy- synthesized methyl a-isomaltoside,

Vmax/Km values for hydrolysis of methyl a-isomaltoside are comparable for the three enzymes, the a-glucosidases from

730 T. P. Frandsen et al.

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Table 1. Specificity constants and DDG(cid:224)a (kJÆmol)1) for a-glucosidase catalyzed hydrolysis of methyl a-isomaltoside and a series of mono-deoxy- genated analogs.

Oligo-1,6-glucosidaseb a-glucosidase (brewer’s yeast)c a-Glucosidase (baker’s yeast)d

DDG(cid:224) Vmax/Km (s)1ÆU)1) DDG(cid:224) Vmax/Km (s)1ÆU)1) DDG(cid:224) Vmax/Km (s)1ÆU)1)

–

– 2.3 15

a DDG(cid:224) (cid:136) )RT ln[(Vmax/Km)a/(Vmax/Km)b] [43], where a and b refer to analog and parent substrate, respectively; b At 30 (cid:176)C using 0.1 M sodium maleate, pH 6.8; c At 30 (cid:176)C using 50 mM phosphate, pH 6.; d At 30 (cid:176)C using 50 mM phosphate, pH 6.8; e standard deviation.

bottom of a cleft between domain B and several of the b fi a connecting segments [14,30]. The molecular recog- nition of isomaltose analogs described above indicate very strong interaction of the nonreducing substrate ring at the enzyme subsite )1, most probably with charged side chains, as a major driving force for stabilization of the enzyme– substrate transition-state. Several of the side-chains inter- acting at subsite )1 will belong to the consensus sequence motifs containing catalytic acids, transition-state stabilizing histidines, and structurally important arginine and aspartate side chains [30].

brewer’s and baker’s yeast showing 31 and 164% of the activity of the oligo-1,6-glucosidase, respectively (Table 1). Furthermore, the activity of the three enzymes was reduced by roughly the same extent, i.e. 440–3400-, 560–2900-, and 1350–8800-fold by substrate deoxygenation at OH-2¢, -3¢, -4¢, or -6¢ (Table 1). The losses in activity compared to the parent substrate for all enzymes were smallest for the 6¢-deoxy analog and largest for the 2¢-deoxy analog, while intermediary losses in activity for 3¢- and 4¢-deoxy analogs did show small variations among the enzymes (Table 1). For the two a-glucosidases, deoxygenation at the reducing end ring of the substrate had no effect or a very minor effect, the activity varying relative to the parent substrate by factors of 0.84–1.4 and 0.42–1.0 for the enzymes from brewer’s and baker’s yeast, respectively. In contrast, for oligo-1,6-glucosidase, -3, and -4 analogs the deoxy-2, showed ninefold, fivefold, and no reduction in Vmax/Km, respectively (Table 1).

While protein–substrate contacts at subsite )1 provide major binding energy, the distribution and strength of intermolecular hydrogen bonds involving the aglycon moiety and subsite +1, as well as subsites beyond subsite +1 in type III a-glucosidases, exhibit substrate specificity variation among the a-glucosidases. The yeast a-glucosid- ases as reported here only show protein–carbohydrate hydrogen bonding involving subsite )1, and no sugar OH-groups associated stabilization energy was critical for accommodation at subsite +1. As shown in Table 2, these a-glucosidases that do not require hydrogen bonding to the

hydrolysis.

substrate

affected

suggesting that

respectively,

The DDG(cid:224) calculated from the Vmax/Km values deter- mined for a given analog and the parent substrate, respectively, indicated the energy contributed to transition- state stabilization by corresponding the OH-group. Because DDG(cid:224) for the four deoxy-analogs at the nonreducing sugar ring, that binds to the enzymes at subsite )1, was in the range 16.1–24.0 kJÆmol)1 for the three enzymes (Table 1), the removal of one of the OH-groups from this ring dramatically These OH-groups can therefore be considered key polar groups and most likely interact with charged residues on the proteins [44] (Fig. 1). At the reducing end ring, however, DDG(cid:224) values of 4–6 kJÆmol)1 for oligo-1,6-glucosidase (Table 1) were obtained by replacement of the OH-2 and -3 groups, these OH-groups participate in neutral hydrogen bonds with the enzyme (Fig. 1). The OH-4 did not seem important in substrate binding and hydrolysis.

Methyl-a-isomaltoside 2-Deoxy-methyl-a-isomaltoside 3-Deoxy-methyl-a-isomaltoside 4-Deoxy-methyl-a-isomaltoside 2¢-Deoxy-methyl-a-isomaltoside 3¢-Deoxy-methyl-a-isomaltoside 4¢-Deoxy-methyl-a-isomaltoside 6¢-Deoxy-methyl-a-isomaltoside 1.4 · 10)4 (cid:139) 0.8 · 10)5e – 1.6 · 10)5 (cid:139) 0.9 · 10)6 2.9 · 10)5 (cid:139) 0.6 · 10)6 1.4 · 10)4 (cid:139) 1.1 · 10)5 4.1 · 10)8 (cid:139) 5.5 · 10)9 1.1 · 10)7 (cid:139) 6.5 · 10)9 1.0 · 10)7 (cid:139) 1.1 · 10)8 3.2 · 10)7 (cid:139) 2.1 · 10)8 5.8 4.1 – 21.5 18.9 19.1 16.1 4.4 · 10)5 (cid:139) 2.9 · 10)6 3.7 · 10)5 (cid:139) 4.9 · 10)6 0.5 6.0 · 10)5 (cid:139) 1.4 · 10)5 )0.8 5.7 · 10)5 (cid:139) 0.8 · 10)6 )0.7 1.5 · 10)8 (cid:139) 1.2 · 10)9 21.2 2.7 · 10)8 (cid:139) 5.3 · 10)9 19.6 1.5 · 10)8 (cid:139) 4.4 · 10)10 21.2 7.9 · 10)8 (cid:139) 4.7 · 10)9 16.7 2.3 · 10)4 (cid:139) 3.2 · 10)6 9.7 · 10)5 (cid:139) 5.8 · 10)6 1.3 · 10)4 (cid:139) 1.3 · 10)5 2.4 · 10)4 (cid:139) 2.0 · 10)6 )0.1 2.6 · 10)8 (cid:139) 3.0 · 10)9 24.0 2.7 · 10)8 (cid:139) 3.1 · 10)9 23.9 3.2 · 10)8 (cid:139) 2.8 · 10)9 23.5 1.4 · 10)7 (cid:139) 1.5 · 10)8 19.6

The three-dimensional structure of oligo-1,6-glucosidase from Bacillus cereus [14], is currently the only available structure of any type of a-glucosidases. This enzyme has an N-terminal (b/a)8 barrel common to glycoside hydrolase family 13 [30,31], a domain B that protrudes from the barrel b strand 3, and a C-terminal Greek key motif. Moreover several extra-barrel secondary structure elements occur in the segments that connect the b strands to the a helices of the (b/a)8 barrel fold [14]. The catalytic site is located at the

Fig. 1. Schematic representation of proposed intermolecular hydrogen bond interactions between isomaltose and a-glucosidases from baker’s and brewer’s yeast and from oligo-1,6-glucosidase from baker’s yeast. a, only for oligo-1,6-glucosidase. Invariant glycoside hydrolase family 13 side chain candidates of interaction with the four nonreducing substrate ring OH-groups are described in detail in a recent review [30].

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Table 2. Kinetic parameters and DDG(cid:224) for hydrolysis of isomaltose and p-nitrophenyl-a-D-glucopyranoside, and mono-deoxy analogs of methyl a-isomaltoside at binding subsite +1 by a-glucosidases and glucoamylase.

)1)

Substrate DDG(cid:224)a (kJÆmol)1) kcat (s)1) Km (mM) kcat/Km (s)1ÆmM

a-Glucosidase (Brewer’s yeast)b 5.2 0.15 135 34.5 0.2 677

Isomaltose p-Nitrophenyl-a-D-glucopyranoside OH-2 OH-3 OH-4 0.5 )0.8 )0.7 Oligo-1,6-glucosidasea 4.8 33.3 129 6.9 1.3 988

5.8 4.1 Isomaltose p-Nitrophenyl-a-D-glucopyranoside OH-2 OH-3 OH-4 –

0.41 0.50 19.8 3.7 0.021 0.135

a Data from Table 1; b [28]; c

Glucoamylasec Isomaltose p-Nitrophenyl-a-D-glucopyranoside OH-2 OH-3 OH-4 1.1 8.6 16.5

(Table 2). The substrate specificity differences and varia- tions in aglycon-protein contacts with the two a-glucosid- ases, the oligo-1,6-glucosidase, and glucoamylase emphasize that these enzymes display different geometry for the binding interactions with polar groups of substrates at subsite +1. This will be investigated further in a study of the diastereo- isomer specificity of isomaltoside hydrolysis (see below).

Catalytic mechanism

One feature of the disposition of substrate relative to enzyme during the various steps of the catalytic events directly relates to the mechanism of catalysis being funda- mentally different for retaining and inverting enzymes [38]. The stereochemistry of isomaltose hydrolysis by yeast oligo- 1,6-glucosidase and a-glucosidases was confirmed to involve retention of the substrate anomeric configuration in the product. This is illustrated for baker’s yeast a-glucosidase which shows 1H NMR spectra of isomaltose before (Fig. 2A) and after (Fig. 2B,C) addition of the enzyme.

substrate aglycon also have much higher activity for p-nitrophenyl-a-D-glucopyranoside, which lacks hydrogen bonding groups in the aglycon, than for isomaltose. Due to effects on both kcat and Km yeast a-glucosidase thus has 4500-fold lower kcat/Km for isomaltose than for p-nitrophe- nyl-a-D-glucopyranoside, p-nitrophenol being also a better leaving group than glucose. Structural elements of the nonsugar aglycon, however, were not explored. It is conceivable, however, that such specificity exists and could be investigated using a series of synthetic substrates. In contrast, the activity of oligo-1,6-glucosidase significantly depends on aglycon interactions at subsite +1 via neutral hydrogen bonds with glucose OH-2 and -3 (Table 1). That such protein interactions with sugar OH-groups are impor- tant for this enzyme in contrast to the two a-glucosidases is also emphasized by the 225-fold difference in the value of the relative specificity p-nitrophenyl a-glucoside/isomaltose, (kcat/Km)/(kcat/Km), being 4500 for the brewer’s a-glucosi- dase (which was chosen because it has the smallest requirement for OH-groups at subsite +1; see Table 1), and 20 for oligo-1,6-glucosidase (Table 2). Moreover, the 30-fold more favorable specificity constant (kcat/Km) for isomaltose for the oligo-1,6-glucosidase over the a-glucosi- dase indeed reflects the genuine specificity of the former enzyme for exo-action on the a-1,6-linkage.

Remarkably, barley a-glucosidase of glycoside hydrolase family 31 has a completely different pattern for hydrolysis of monodeoxy maltoside analogs which indicated strong protein–substrate interactions at OH-4¢ and -6¢ and weaker, probably neutral hydrogen-bonds with maltose OH-2¢, -3¢, and -3 [22]. An even stronger requirement for protein– isomaltose aglycon interactions was found in isomaltose hydrolysis by glucoamylase, which depended on enzyme– substrate transition-state interactions with OH-4 and -3 of an energy of 16.5 and 8.6 kJÆmol)1, respectively (Table 2; [7]). Glucoamylase thus has only sixfold lower kcat/Km for isomaltose than for p-nitrophenyl-a-D-glucopyranoside

[7,51].

Fig. 2. Hydrolysis of isomaltose by baker’s yeast a-glucosidase followed by 1H NMR. (A) before addition of enzyme; (B) 4 min; and (C) 16 h after addition of enzyme.

732 T. P. Frandsen et al.

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Recognition of diastereoisomeric isomaltoside derivatives

Comparison of these spectra showed the appearance of a doublet centered at 5.22 p.p.m. This was assigned to H-1 of free a-glucose while the resonance at 4.64 p.p.m., which appeared later was assigned to H-1 of b-glucose which the initially released stemmed from mutarotation of a-glucose. The anomer ratio of D-glucose (33% a: 67% b) was deduced from the 1H NMR spectrum after complete hydrolysis of isomaltose (Fig. 2C) and falls within the range normally found for the equilibrium mixture. The stereo- chemistry of the products thus confirmed that the three a-glucosidases catalyze hydrolysis of isomaltose with reten- tion of the anomeric configuration as is characteristic of family 13 glycoside hydrolases. Figure 3 shows the widely accepted double displacement mechanism for retaining hydrolases, which is believed to occur through oxacarbeni- um ion transition-states and formation of a covalent intermediate between the catalytic nucleophile and the C-1 of the substrate glycon [18,30,31,45–49]. Further kinetics analyses are not feasible due to the limited amounts of analogs available; we therefore cannot determine the role of a key polar group in the glycosylation or the deglycosylation steps in the mechanism (Fig. 3). However, one can conclude that the discrimination of the diastereoisomer, as this is associated with the Vmax and not the Km, does not happen in the initial reversible part of substrate complex formation, but in subsequent steps of the catalytic mechanism [40].

Isomaltose is flexible due to rotation around the C5–C6 bond. It is possible to block this conformational flexibility by alkylation of C6 (Fig. 4). Previously, methyl 6-R- and methyl 6-S-methyl-a-isomaltoside were used to determine the preferred rotational conformer for glycoamylase [40]. Hydrolysis catalyzed by baker’s yeast a-glucosidase (this enzyme was chosen as it has the highest activity of the two a-glucosidases; see Table 1) was similarly examined using methyl 6-R-ethyl- and methyl 6-S-ethyl-a-isomaltoside as the pair of conformationally biased substrate analogs (Table 3). While methyl 6-S-ethyl-a-isomaltoside was hydrolyzed with twofold lower Vmax, but the same Km as isomaltose (Table 3), the 6-R enantiomer was a poor substrate Vmax being 150-fold lower and Km twofold higher than for isomaltose (Table 3). Baker’s yeast a-glucosidase thus preferred the 6-S isomer. In contrast, glucoamylase from A. niger hydrolyzed the 6-R enantiomer with 230-fold higher kcat/Km compared to the parent isomaltoside, the difference being essentially in the Km and not in the rate of hydrolysis as for the a-glucosidase [40]. This distinct preference for one of the two diastereoisomers of the C-6 alkyl isomaltose derivatives reflects the fact that one of the rotamers adopts a conformation with more favorable

Fig. 3. Catalytic mechanism for retaining gly- coside hydrolases including steps of protonation, formation of a covalent intermediate, and product release, respectively, but not the inter- mediate two transition-states (see text for details and [30,31,39]).

A

B

Fig. 4. Structure of the conformationally biased diastereosisomer substrates methyl 6-R- ethyl-a-isomaltoside (A) and methyl 6-S-ethyl- a-isomaltoside (B).

Table 3. Kinetic parameters for the hydrolysis of conformationally biased isomaltosides.

Substrate Vmax (mMÆ s)1ÆU)1) Km (mM) Vmax/Km (s)1ÆU)1)

a-Glucosidase from baker’s yeasta

)1)

Isomaltose Methyl 6-S-ethyl-a-isomaltoside Methyl 6-R-ethyl-a-isomaltoside 2.8 · 10)3 1.6 · 10)3 1.8 · 10)5 9.8 9.6 19.4 2.8 · 10)4 1.7 · 10)4 9.3 · 10)7

a At 30 (cid:176)C, using 50 mM phosphate, pH 6.8. b [40].

Glucoamylase from A. nigerb Methyl a-isomaltoside Methyl 6-S-methyl-a-isomaltoside Methyl 6-R-methyl-a-isomaltoside kcat (s)1) 1.04 1.1 0.68 Km (mM) 24.5 90.0 0.71 kcat/Km (s)1ÆmM 0.0042 0.012 0.96

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A C K N O W L E D G E M E N T S

Bent O. Petersen is gratefully acknowledged for performing the NMR spectroscopy experiments and Ulrike Spohr and Raymond U. Lemieux are thanked for the synthetic substrate analogs. This work was supported in part by funding from the Natural Sciences and Engineering Research Council of Canada (to M. M. P).

R E F E R E N C E S

spatial distribution of the groups that play an important role in the enzyme recognition. This finding stresses the fundamentally different active site architecture that exists for the inverting glucoamylase and the retaining a-gluco- sidases. Glucoamylase, in contrast to a-glucosidase, applies a single displacement mechanism and belongs to a different fold family, glycoside hydrolase 15. The specific activities and substrate affinities are similar for these retaining and inverting enzymes, all of which have reasonable capacity in the glucose release from the nonreducing end of disac- charides and small substrates. However, the a-glucosidase showed large variation in rate of hydrolysis between the methyl 6-S- and 6-R-ethyl a-isomaltosides, with small differences in affinity for the two distereoisomers, whereas the discrimination by glucoamylase was associated with the Km [40] and not with the rate of hydrolysis (Table 3).

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C O N C L U S I O N

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The enzyme preparations used in the present analysis are considered valuable representatives of two categories of yeast a-glucosidases. The study strongly demonstrates the advantage offered by enzymes with simple specificity for application of substrate analogs in elucidation of the roles of individual substrate groups or atoms in binding and catalysis. This is in contrast to other enzymes of the glycoside hydrolase family 13 catalysing polysaccharide degradation in an endo-like fashion, for which even model substrates would typically be rather large and hence extremely difficult, laborious and costly to synthesize. In addition, the option of several functional binding modes in the active site cleft in these latter enzymes obscures interpretation using analogs of the impact of specific substrate groups on catalysis. The fact that the enzymes used in the present study possess a simple substrate specificity and belong to the large family 13 representing 28 substrate specificities [30] suggests an application of the present findings, ultimately, for rational protein engineering of these and other family members with other specificities. Contacts with invariant Arg, His, and Asp residues involved in charged hydrogen bonds to the glucose ring at subsite )1 in family 13 (reviewed in [30]) are thus proposed to be responsible for the reported major role of hydroxyl groups of this ring in transition-state stabilization. While the invariant Asp plays a role in catalysis [47] and mutation leads to inactivation, the single mutation to Asn of each of two His interacting at subsite )1 with OH-6 and OH-2 plus OH-3, in case of barley a-amylase 1, affected transition- state stabilization and reduced activity to 5 and 10%, respectively [50]. Structure guided sequence comparisons, in contrast, do not yet allow tentative identification of specific residues that are important for the interactions with the substrate ring at subsite +1 in the oligo-1,6-glucosidase as well as for controlling the exo-action at the level of the nonreducing end ring at subsite )1 of all three a-glucosid- ases included in the present comparison. The findings on substrate key polar groups and preferred isomaltoside diasteroisomers, however, will be valuable in future mod- eling of substrate complexes of the a-glucosidases and related enzymes if the structures become available. The data may thus guide protein engineering studies that address the a-1,4 and a-1,6 bond specificity of these closely related a-glycosidases.

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