The role of residues R97 and Y331 in modulating the pH optimum of an insect b-glycosidase of family 1 Sandro R. Marana, Lu ´ cio M. F. Mendonc¸a, Eduardo H. P. Andrade, Walter R. Terra and Cle ´ lia Ferreira Departamento de Bioquı ´ mica, Instituto de Quı ´ mica, Universidade de Sa ˜ o Paulo, Brazil The activity of the digestive b-glycosidase from Spodoptera frugiperda (Sfbgly50, pH optimum 6.2) depends on E399 (pK a ¼ 4.9; catalytic nucleophile) and E187 (pK a ¼ 7.5; catalytic proton donor). Homology modelling of the Sfbgly50 active site confirms that R97 and Y331 form hydrogen bonds with E399. Site-directed mutagenesis showed that the substitution of R97 by methionine or lysine increased the E399 pK a by 0.6 or 0.8 units, respectively, shifting the pH optima of these mutants to 6.5. The substi- tution of Y331 by phenylalanine increased the pK a of E399 and E187 by 0.7 and 1.6 units, respectively, and displaced the pH optimum to 7.0. From the observed DpK a it was calcu- lated that R97 and Y331 contribute 3.4 and 4.0 kJÆmol )1 , respectively, to stabilization of the charged E399, thus enabling it to be the catalytic nucleophile. The substitution of E187byDdecreasedthepK a of residue 187 by 0.5 units and shifted the pH optimum to 5.8, suggesting that an electro- static repulsion between the deprotonated E399 and E187 may increase the pK a of E187, which then becomes the catalytic proton donor. In short the data showed that a network of noncovalent interactions among R97, Y331, E399 and E187 controls the Sfbgly50 pH optimum. As those residues are conserved among the family 1 b-glycosidases, it is proposed here that similar interactions modulate the pH optimum of all family 1 b-glycosidases. Keywords: b-glycosidase; pK a values; pH optimum; site- directed mutagenesis; Spodoptera frugiperda. The b-glycosidases from glycoside hydrolase family 1 are enzymes that remove monosaccharides from the nonreduc- ing end of di- and/or oligosaccharides. According to the CAZy website this family comprises 422 sequenced b-glycosidases, of which the tertiary structure of 12 has been determined. Together with families 2, 10, 17, 26, 30, 35, 39, 42, 51, 53, 59, 72, 79 and 86 family 1 forms clan A, a group of families that shares structural and catalytic similarities [1]. All b-glycosidases of family 1 present the same tertiary structure [the (b/a) 8 barrel], they are configur- ation-retaining glycosidases and their catalytic activity depends on two glutamic acid residues, one positioned after bstrand 4 and the other after b strand 7 [1]. These glutamic acids are very close inside the active site (about 4.5 A ˚ apart) [2], and during the reaction the first glutamic acid acts as proton donor, and the second acts as a nucleophile. The catalytic nucleophile pK a is around 5.0 and the catalytic proton donor pK a is around 7.0 [3–7]. Aplotofb-glycosidase activity vs. pH presents a bell shape, indicating that in the pH optimum the catalytic nucleophile is deprotonated and the catalytic proton donor is protonated. Hence the branch of the curve below the pH optimum is determined mainly by the ionization of the catalytic nucleophile, whereas the catalytic ionization of the proton donor determines the branch above the pH optimum. As the b-glycosidase activity depends on the finely tuned ionization of the catalytic nucleophile and proton donor, it is necessary to understand how the ionization of these residues is modulated in order to determine how the pH optimum is controlled. Such data are lacking for family 1. In family 11 xylanases, it was proposed that the negatively charged nucleophile electrostatically destabilizes the proton donor ionization, increasing its pK a [8]. Thus, one may hypothesize that the Ôelectrostatic couplingÕ between the catalytic glutamates could also operate in family 1, despite the fact that the family 11 and 1 belong to different clans. However, even this interaction is not enough to determine which of the glutamic acid residues will be the catalytic nucleophile or the proton donor. This is because for b-glycosidases it is not known how the ionization of each catalytic glutamate is modulated and therefore there is no model to explain how the pH optimum is determined. In this work a digestive b-glycosidase from Spodoptera frugiperda (Sfbgly50) was used as an experimental model to fill those gaps. This enzyme had been previously sequenced (GenBank accession number AF052729) and it was classi- fied in the glycoside hydrolase family 1. Catalytic activity of Sfbgly50 depends on residues E399 (catalytic nucleophile, pK a ¼ 4.9) and E187 (catalytic proton donor, pK a ¼ 7.5). Correspondence to S. R. Marana, Departamento de Bioquı ´ mica, Instituto de Quı ´ mica, USP, CP 26077, Sa ˜ o Paulo, 05513–970, Brazil. Fax: +55 11 30912186, E-mail: srmarana@iq.usp.br Abbreviations: MU, 4-methylumbelliferyl; MUbglc, 4-methylumbel- liferyl b- D -glucopyranoside; NPbglc, p-nitrophenyl, b- D -gluco- pyranoside; Sfbgly50, digestive b-glycosidase (Mr 50 000) from Spodoptera frugiperda; ES, enzyme substrate. Enzyme:digestiveb-glycosidase from Spodoptera frugiperda (b- D -glucoside glucohydrolase; EC 3.2.1.21; GenBank accession no. AF052729). (Received 18 July 2003, revised 25 September 2003, accepted 21 October 2003) Eur. J. Biochem. 270, 4866–4875 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03887.x The effect of pH on Sfbgly50 activity is a typical bell-shaped curve and the pH optimum is 6.2 [9]. In the active site of the family 1 b-glycosidases the catalytic nucleophile and proton donor are very close (4.5 A ˚ apart). Hence differences in their ionization and resulting catalytic roles should rely on noncovalent inter- actions that are specific for each residue. Structural data on different family 1 b-glycosidases showed out that the catalytic nucleophile interacts with an arginine and a tyrosine, putatively being stabilized by them [10–13]. Homology modelling the Sfbgly50 active site confirms that Y331 and R97 are positioned very close to E399 (less than 3 A ˚ apart), suggesting that these residues are hydrogen bonded with the catalytic nucleophile (Fig. 1). Therefore, these interactions may affect the E399 ioniza- tion so that this residue becomes the catalytic nucleophile, whereas an electrostatic repulsion between E399 and E187 cause this last one to function as the catalytic proton donor. Thus, interactions between E399 and Y331 or R97 may be key elements in the determination of Sfbgly50 pH optimum. The role of arginine in the modulation of the b-glycosidase pH optimum had not been studied before. The substitution of Y298 for phenylalanine in Agrobacte- rium sp. b-glucosidase affected the rate constant of the glycosylation step and also changed the enzyme pH optimum [14]. Despite that, the effect of the tyrosine on the pK a values of the b-glycosidase catalytic glutamates still remains to be determined and quantified. To fill these gaps, residues Y331, R97 and E187 of Sfbgly50 were replaced through site-directed mutagenesis by phenylalanine (Y331F), methionine (R97M), lysine (R97K) and aspartate (E187D). The effect of pH on the activity of the recombinant enzymes were then determined. Materials and methods Materials All reagents, unless otherwise specified, were purchased from Sigma or Merck. Site-directed mutagenesis Site-directed mutagenesis was performed using as template the plasmid pT7-7 [15] containing as insert a DNA fragment that encodes the mature Sfbgly50 (pT7b50) [9]. The experiments were carried out following the instructions of the QuikChange site-directed mutagenesis kit (Stratagene). Primers used were: R97K, 5¢-GCCTGGACGCTTACA AGTTCTCCCTCTCCTG-3¢ and 5¢-CAGGAGAGGGA GAACTTGTAAGCGTCCAGGC-3¢; R97M, 5¢-GCCTG GACGCTTACATGTTCTCCCTCTCCTG-3¢ and 5¢-CA GGAGAGGGAGAACATGTAAGCGTCCAGGC-3¢; Y331F, 5¢-GATCGGAGTGAACCACTTCACAGCATT CCTGGTATC-3¢ and 5¢-GATACCAGGAATGCTGTG AAGTGGTTCACTCCGATC-3¢; E187D, 5¢-GTTCATC ACTTTCAACGATCCTAGAGAGATTTGCTTTGAG-3¢ and 5¢-CTCAAAGCAAATCTCTCTAGGATCGTTGA AAGTGATGAAC-3¢. Codons in bold show the mutations. The incorporation of the mutated codon in the pT7b50 was checked through DNA sequencing. Expression of recombinant Sfbgly50 NovablueDE3 (Novagen) cells were cotransformed with pT7b50 (encoding wild-type or mutant type Sfbgly50) and pT-GroE, a plasmid encoding the chaperone GroELS under the control of the T7 RNA polymerase promoter. pT-GroE increases the Gro-ELS concentration inside the cells and consequently it favours the folding of coexpressed proteins [16]. The transformed bacteria were cultivated (37 °C, 250 r.p.m.) in Luria–Bertani broth containing carbenicillin (50 lgÆmL )1 ) and chloramphenicol (17 lgÆmL )1 ) until D 600 ¼ 0.6–1.0 was reached. The bac- teria were then induced using 1 m M isopropyl thio-b- D -galactoside for 3 h (25 °C, 250 r.p.m.) and harvested at 7000 g for 20 min at 4 °C. The pellets were stored at )80 °C. Samples of induced and noninduced cells were analysed by SDS/PAGE [17] to detect the expression of the recombinant b-glycosidases. Lysis of induced bacteria Induced bacteria were suspended in 50 m M Hepes buffer pH 7.0 containing 150 m M NaCl, 0.02% (w/v) lysozyme (chicken egg white) and 0.1% (v/v) Triton X-100. The suspension was incubated at 4 °C with slow shaking (3 r.p.m.). After 30 min, the cells in the suspension were disrupted using a sonicator adapted with a micro tip (five pulses of 30 s at output 4 in a Branson 250 sonicator) and Fig. 1. Schematic representation of the Sfbgly50 active site. E399 is the nucleophile and E187 is the proton donor. Y331 and R97 form hydrogen bonds (dotted lines) with E399 (Y331 O g atom to E399 O e1 atom ¼ 2.69 A ˚ and R97 N g1 atom to E399 O e2 atom ¼ 2.77 A ˚ ). The distance between E399 and E187 side chains is 4.5 A ˚ . Ó FEBS 2003 Mechanism of pH optimum control in b-glycosidases (Eur. J. Biochem. 270) 4867 harvested at 7000 g for 20 min at 4 °C. The supernatant was stored at )20 °C and used as source of recombinant b-glycosidase. Purification of the recombinant Sfbgly50 Soluble material from the induced cells containing the wild- type or mutant recombinant Sfbgly50 was loaded onto a phenylSuperose HR 10/10 column (Pharmacia Biotech). The nonretained proteins were washed out with 50 m M phosphate buffer pH 7.0 containing 1.27 M (NH 4 ) 2 SO 4 ,and the retained proteins were then eluted using a gradient of (NH 4 ) 2 SO 4 prepared in 50 m M phosphate buffer pH 7.0. ThepresenceoftherecombinantSfbgly50 was detected by enzymatic assay using NPbglc (p-nitrophenyl b- D -glucopyr- anoside) as substrate [18]. Fractions containing b-glycosi- dase activity were pooled and dialysed in 20 m M triethanolamine buffer pH 8.0, and the dialysed material was loaded onto a Resource Q column (Amersham Bio- science). Nonretained proteins were washed out with 20 m M triethanolamine buffer pH 8.0 and retained proteins were eluted using a gradient of NaCl prepared in the same initial buffer. The presence of the recombinant Sfbgly50 was detected as above and its purity ascertained by SDS/PAGE followed by silver staining [19]. Protein determinations were performed spectrophotomet- rically (absorbance in 280 nm) using e 280 ¼ 117 200 M )1 Æcm )1 [20]. The same protocol was used to purify the wild-type and mutant Sfbgly50. The native Sfbgly50 was purified from the S. frugiperda midgut following the procedures described previously [21]. Kinetic analysis All assays were performed at 30 °Cin50m M citrate/ phosphate buffer pH 6.0 and initial rate data measured. Hydrolysis of MUbglc (4-methylumbelliferyl b- D -glucopyr- anoside) was followed by MU fluorescence [22]. Kinetic parameters (k cat and K m ) were determined by using nine different substrate concentrations (0.1–8 m M ); enzyme concentrations were 0.13 l M for R97M, 0.09 l M for Y331F and 0.62 l M for E187D. The data were fitted to Michaelis–Menten equation using the ENZFITTER (Elsevier- Biosoft). Chemical modification with phenylglyoxal Arginine modification was performed using different concentrations of phenylglyoxal (1, 3, 4 and 5 m M ) prepared in 20 m M phosphate buffer pH 8 at 30 °C. In this pH phenylglyoxal reacts specifically with arginine residues [23,24]. Wild-type (0.49 l M )ormutantSfbgly50 (0.13 l M ) samples were incubated with the modifying agent in the absence or presence of high concentration of NPbglc (> 4 · K m ). Samples were collected after differ- ent periods of time and 10 m M arginine in 20 m M phosphate pH 8.0 was added. This material was used to determine the remaining enzymatic activity using 4 m M MUbglc as substrate in 50 m M citrate/phosphate buffer pH 6.0. Then, the rate constants (k obs )fortheSfbgly50 inactivation in different phenylglyoxal concentrations were calculated. pH effect on the Sfbgly50 activity Sfbgly50 enzymatic activity on 8 m M MUbglc was deter- mined in different buffers ranging from pH 5.0 to 8.5 (50 m M citrate/phosphate buffer, pH 5.0–7.0; 50 m M phos- phate buffer, pH 7.0–8.0; 50 m M Bicine buffer 7.0–8.5). The pH stability of Sfbgly50 was checked by incubation in the same buffers for a time equal to the assay time and then determining the activity remaining at the optimum pH. To correct the pH shifts due to differences in temperature, pH of the assay media was measured in substrate/buffer mixtures at 30 °C. The enzyme concentration was 0.13 l M for mutant R97M, 0.09 l M for mutant Y331F and 0.37 l M for mutant E187D. At 8 m M MUbglc, Sfbgly50 is approaching saturation by the substrate. Hence, relative activity is a good approxima- tion of the relative maximum velocity (V max app ) under these conditions. Thus the pK a s in the enzyme–substrate (ES) complex of the catalytically active groups of Sfbgly50 (pK ES1 and pK ES2 ) were determined by fitting the V max app of the MUbglc hydrolysis at each pH to Eqn (1) [25]. V max app ¼ 1 1 þ H þ ÂÃ K ES1 þ K ES2 H þ ÂÃ  ð1Þ V max app is the relative V max determined at each pH, [H + ] is the proton concentration and K ES1 and K ES2 are the ionization constants of the two catalytically essential groups in the ES complex. V max app was expressed as a percentage of the highest V max observed, and fitting was done using the software ENZFITTER . Ionization constants in the free enzyme (pK E1 and pK E2 ) were calculated using Eqn (2) [25]. k cat =K mapp ¼ 1 1 þ H þ ÂÃ K E1  þ K E2 H þ ÂÃ  ð2Þ k cat /K mapp is the relative k cat /K m determined at each pH, [H + ] is the proton concentration and K E1 and K E2 are the ionization constants of the two catalytically essential groups in the free enzyme. k cat /K mapp may be calculated from the enzymatic activity determined using low substrate concentration (0.25 m M ) at different pH values and relative k cat /K mapp and [H + ] were fitted in the above equation using ENZFITTER . The data were enough to fit simultaneously the two K ES and K E . In the E187D mutant, the determination of pK ES1 was less accurate than that of pK ES2 , because many more points above the pH optimum were obtained. However, it was not possible to go below pH 5.0 because Sfbgly50 becomes unstable. Homology modelling The three-dimensional structure of Sfbgly50 was modelled according to structural data for Bacillus polymyxa b-glucosidase A (1BGA, 1BGG, 1TR1), Trifolium repens b-glucosidase 2 (1CBG) and Lactococcus lactis 6-phospho b-galactosidase (1PBG). Modelling was performed in the Swiss Model server and the result was visualized by PDBVIEWER [26]. 4868 S. R. Marana et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Sequence alignment and structural comparison Amino acid sequences of family 1 b-glycosidases were retrieved from the CAZy website [1] and aligned using the software CLUSTALX [27]. The spatial coordinates of family 1 b-glycosidases were retrieved from the PDB website and visualized by PDBVIEWER [26]. Results The expression of recombinant wild-type and mutant Sfbgly50 was checked by SDS/PAGE (Fig. 2A). Recom- binant Sfbgly50 was purified by a combination of hydro- phobic and anion-exchange chromatography (Fig. 2B), resulting in a 50% recovery and a yield of about 0.2 mg purified Sfbgly50 from 0.5 L bacterial culture (Fig. 2C). The kinetic parameters (k cat and K m ) for the hydrolysis of MUbglc were determined for the Sfbgly50 mutants (R97M, Y331F and E187D) and compared with those of the wild- type enzyme (Table 1). All mutations resulted in a large activity decrease, indicating that R97 and Y331 do influence catalysis. The pH-dependent activity profile is similar for the native and recombinant wild-type Sfbgly50 (Fig. 3). Hence the recombinant wild-type Sfbgly50 is useful as a control in comparisons with the Sfbgly50 mutants. Moreover, wild- type and mutant Sfbgly50 are stable in the pH range 5.0–9.0 (Fig. 3). As the kinetic data showed that R97 and Y331 influence catalysis, the function of residue R97 was investigated by performing a chemical inactivation of wild-type Sfbgly50 with phenylglyoxal. The reaction order was 1.7 in relation to phenylglyoxal and the inactivation was halted by the presence of saturating concentrations of substrate (Fig. 4). In contrast, phenylglyoxal did not inactivate R97M and R97K mutants (data not shown). Enzyme activity–pH data showed that R97K and R97M Sfbgly50 mutants presented curves narrower and pH optimum (6.5) slightly higher than wild-type Sfbgly50 (6.2) (Fig. 5A,B). The mutant Y331F presented an activity–pH profile wider and a pH optimum (7.0) higher than the wild- type Sfbgly50, whereas the E187D mutant had a pH optimum (5.8) lower than the recombinant wild-type enzyme (Fig. 5C,D). Fig. 2. Induction and purification of the recombinant Sfbgly50. (A) SDS/PAGE of proteins from NovablueDE3 cells transformed with plasmid pT7-7 containing Sfbgly50. Lane 1, Noninduced cells; lanes 2, 3, 4 and 5, cells induced to produce the mutants R97M, R97K, Y331F and E187D, respectively. The arrow indicates the recombinant Sfbgly50. The gel (10% polyacrylamide) was stained with Coomassie blue R. (B) The soluble material from the bacteria producing the R97M Sfbgly50 was loaded onto a Phenyl Superose HR 10/10 column eluted with a decreasing gradient of (NH 4 ) 2 SO 4 ,preparedin50m M phosphate buffer pH 7.0. b-Glycosidase activity (r) was detected using 2 m M NPbglc prepared in 50 m M citrate/phosphate buffer pH 6.0. The two most active fractions were pooled. (C) Ion-exchange chromatography in Resource Q column of the b-glycosidase activity recovered in (B). Elution produced using a gradient of NaCl prepared in 20 m M triethanolamine buffer pH 8.0. b-Glycosidase activity (r) was detected using NPbglc. The three most active fractions were pooled and analysed by SDS/PAGE. (D) SDS/PAGE of the purified Sfbgly50. The gel (10% polyacrylamide) was silver stained. The same procedure was used to purify the wild-type and mutant (R97K, Y331F and E187D) Sfbgly50. As the gels are not the same size the band positions are not directly comparable. Ó FEBS 2003 Mechanism of pH optimum control in b-glycosidases (Eur. J. Biochem. 270) 4869 As the pK a values of the catalytic residues determine the pH optima, the ionization constants (pK a )ofthecatalytic glutamates (E187 and E399) in the ES complex and in the free enzyme (E) of Sfbgly50 mutants were determined. The pK ES and pK E are very similar, thus only the pK ES values are presented (Table 2). The differences observed in pK a values between the wild-type and mutant enzymes result from the modifications in the interactions between the catalytic glutamates and the residues R97 and Y331, indicating that these residues play a role in the modulation of the Sfbgly50 pH optimum. Sequence alignments and structural comparisons of family 1 b-glycosidases showed that R97 and Y331 are totally conserved and that these residues plus the nucleo- phile (E399) have the same spatial positioning (Fig. 6). Nevertheless, the distance between arginine and glutamate varies from 2.59 to 3.64 A ˚ and the distance between the tyrosine and glutamate varies from 2.59 to 2.98 A ˚ . Discussion An inspection of the three-dimensional structure of some family 1 b-glycosidases [10–13] and of the structural model of the Sfbgly50 active site show that an arginine (R97) and a tyrosine (Y331) are very close (2.69 A ˚ and 2.77 A ˚ , respect- ively) and form hydrogen bonds with the side chain of E399. The hydrogen bond between R97 and E399 probably has a strong electrostatic component. However, determination of the relative contribution of each component in this inter- action is not simple. On the other hand, E399 is also close to E187 side chain (4.5 A ˚ ) and these residues may interact electrostatically (Fig. 1). These noncovalent interactions may modulate the E187 and E399 ionization state and consequently determine the Sfbgly50 pH optimum. In order to test this hypothesis, some mutants (R97M, R97K, Y331F and E187D) were expressed as recombinant proteins in Escherichia coli. The general shape and volume of residues 97 and 331 side chains are conserved in the mutants R97M and Y331F, but the hydrogen bond-forming atoms have been removed. In mutant E187D, the distance between the catalytic nucleophile and the catalytic proton donor has been increased because the side chain of aspartic acid is shorter than that of glutamic acid. The kinetic parameters for MUbglc hydrolysis (Table 1) show that the substitution of R97 and Y331 results mostly in a decrease in k cat ,whereasK m is affected less, suggesting that these residues influence catalysis. As a member of the glycoside hydrolase family 1, the Sfbgly50 probably follows a double displacement mechanism with a glycosyl-enzyme intermediate. However, as it is not known which step (glycosylation or deglycosylation) of the hydrolysis of MUbglc by the mutant enzymes is rate-limiting, no hypo- thesis on the effect of R97 and Y331 on the rate constant of each step can be advanced. Table 1. Steady-state kinetic parameters for hydrolysis of MUbglucoside by recombinant wild-type and mutant Sfbgly50. The experiments were carried out at nine different substrate concentrations and the parameters were calculated using ENZFITTER . K m (m M ) k cat (s )1 ) k cat /K m (s )1 Æm M )1 ) k cat /K m relative Wild-type 2.3 ± 0.1 1.73 ± 0.09 0.75 ± 0.06 100 R97M 1.9 ± 0.3 0.0030 ± 0.0002 0.0015 ± 0.0005 0.2 Y331F 2.0 ± 0.5 0.0070 ± 0.0005 0.003 ± 0.001 0.45 E187D 4.4 ± 0.1 0.00147 ± 0.00002 0.00033 ± 0.00001 0.044 Fig. 3. Effect of pH on the activity of native (j)andrecombinant(s) wild-type Sfbgly50. The buffers used were 50 m M citrate/phosphate (pH 4.7–7.0), 50 m M phosphate (pH 7.0–8.0) and 50 m M bicine (pH 8.0–8.5). Each point is the average of four Sfbgly50 activity determinations using 4 m M MUbglc as substrate. The pH stability of the recombinant Sfbgly50 (n) was checked by incubating the enzyme in the same buffers for an equal length of time and determining the remaining activity in the pH optimum. Fig. 4. Inactivation of the Sfbgly50 with phenylglyoxal. Effect of phenylglyoxal concentration (r,1m M ; j,3m M ; m,4m M ; d,5m M ) on the inactivation rate of the wild-type recombinant Sfbgly50. Phe- nylglyoxal was prepared in 20 m M phosphate buffer pH 8.0. The inactivation order is 1.7 with phenylglyoxal as calculated from the insert. Enzymatic activity was detected using as substrate 4 m M MUbglc in 50 m M citrate/phosphate pH 6.0. 4870 S. R. Marana et al. (Eur. J. Biochem. 270) Ó FEBS 2003 The influence of R97 on catalysis is confirmed by the phenylglyoxal inactivation (Fig. 4), which is abolished by substrate and is not observed with the R97M and R97K mutants. The reaction order relative to phenylglyoxal (1.7) indicates that the enzyme is inactivated by the reaction of two phenylglyoxal molecules with one arginine residue. Despite the fact that the reaction mechanism is not clear (a dimer or two phenylglyoxal molecules may react with one arginine residue), the reaction order (1.7) is in agreement with the proportion found in reactions between phenyl- glyoxal and polypeptides (2 : 1) [23]. The structure of the putative reaction product [23] indicates that the modified R97 side chain is unable to hydrogen bond with E399, probably causing wild-type Sfbgly50 inactivation. More- over, the addition of a bulky group (diphenylglyoxal) in the active site probably hinders substrate binding. The substitution of R97 by M results in a 500-fold decrease in k cat , whereas the replacement of Y331 by F results in a 250-fold decrease (compare k cat for the wild-type and mutant Sfbgly50 in Table 1). As the extent of k cat decrease is similar in both cases, R97 and Y331 may have a similar influence on catalysis. In a b-glycosidase from Agrobacterium sp. (glycoside hydrolase family 1), the replacement of the residue equivalent to Y331 (Y298, Agrobacterium numbering) for a phenylalanine also results in a large k cat decrease (500-fold) [14]. One possible effect of R97 and Y331 on catalysis is to position E399. Thus, the k cat decrease could result from an incorrect positioning of the catalytic nucleophile, but the data presented here are not enough to support this hypothesis. In the case of the Y331F mutant part of the decrease in k cat may result from destabilization of the ES complex, because the tyrosine residue is thought to interact with the oxygen of the glycone ring in that complex [10,12]. Nevertheless, it is not possible to assume that the same is occurring in the mutant R97M, because there is no data on the interactions between the substrate and the arginine residue. Finally, another aspect of the R97 and Y331 influence on catalysis that must be taken into account is the modulation of the ionization state of the catalytic glutamates. Indeed, the substitution of R97 by M, which disrupts the hydrogen bond between residue 97 and E399, shifted the E399 pK a by +0.6 pH units (from 4.8 to 5.4), but had no effect on the E187 pK a . As a consequence of the higher pK a of E399, the mutant R97M has a pH optimum (6.5) slightly higher than that of wild-type Sfbgly50 (6.2). Although the introduction of a methionine residue at position 97 could have changed the dielectric constant of the active site, the deletion of the hydrogen bond between R97 and E399 is probably a major cause of the shift in the pK a of E399. Therefore, R97 facilitates the ionization of the catalytic nucleophile by stabilizing its charged state. Fig. 5. Effect of pH on the relative maximum velocity (V maxapp )ofthe wild-type (s) and mutant Sfbgly50 (j). (A) R97K; (B) R97M; (C) Y331F; (D) E187D. The buffers used were 50 m M citrate/phosphate (pH 4.7–7.0), 50 m M phosphate (pH 7.0–8.0) and 50 m M bicine (pH 8.0–8.5). Each point is the average of four Sfbgly50 activity determinations using 8 m M MUbglc as substrate. The enzymes are stable in this pH range. Based on these data, pK ES1 and pK ES2 values were calculated as described in the Material and methods. Table 2. pK a values of the catalytic groups in the ES complex of the wild-type and mutants Sfbgly50. pK ES1 pK ES2 DpK ES1 DpK ES2 Wild-type 4.8 ± 0.1 7.4 ± 0.1 – – R97K 5.6 ± 0.1 7.7 ± 0.1 +0.8 +0.3 R97M 5.4 ± 0.1 7.5 ± 0.1 +0.6 +0.1 Y331F 5.5 ± 0.1 9.0 ± 0.1 +0.7 +1.6 E187D 4.5 ± 0.1 6.9 ± 0.1 )0.3 )0.5 Ó FEBS 2003 Mechanism of pH optimum control in b-glycosidases (Eur. J. Biochem. 270) 4871 The observed DpK a are directly related to the differences in the free energy change (DDG° ¼ 2.303RTDpK a )of ionization of the groups in wild-type and mutant enzyme. This ionization differs because of the stabilizing effect provided by R97, which is lacking in the mutant R97M. Hence, the DDG° is equal to the energy of the stabilizing effect provided by R97. Thus, based on the DpK a of E399 between the wild-type and R97M Sfbgly50, it was calculated that R97 contributes 3.4 ± 0.4 kJÆmol )1 to stabilize the charge of E399. In the R97K mutant the pK a of E399 is shifted by +0.8 pH units, whereas pK a of E187 changed by +0.3 pH Fig. 6. Sequence alignment and structural comparison of family 1 glycoside hydrolases. (A) Sequence alignment of the regions containing the residue R97 and Y331 (Sfbgly50 numbering). The aligned b-glycosidases are from Actinomyces naeslundii AAK33123.1, Agrobacterium sp. AAA220851, Arabidopsis thaliana Q9SE50, Bacillus circulans Q03506, Bacillus polymyxa P22073, Brassica napus Q42618, Catharanthus roseus AAF28800.1, Cavia porcellus P97265, Clostridium longisporum Q46130, Escherichia coli K12 P11988, Lactobacillus caseii P14696, Lactococcus lactis P11546, Prunus serotina AAL06338.1, Pyrococcus woesei O52626, Sinapis alba P29092, Spodoptera frugiperda AF052729, Staphylococcus aureus P11175, Sulfolobus solfataricus P22498, Thermus nonproteolyticus AAF36392.1, Trifolium repens P26205, Zea mays P49235. An asterisk marks identical residues, a colon indicates strongly conserved residues and a period denotes weakly conserved residues. (B) The spatial position of the residues corresponding to Y331, E399 and R97 (Sfbgly50 numbering) in different glycoside hydrolases was superimposed. The spatial coordinates were retrieved from PDB: 1BGG, b-glycosidase from Bacillus polymyxa (black; R77, Y296 and E352); 2MYR, myrosinase from Sinapis alba (green; R95, Y330 and E409); 1CBG, cyanogenic b-glycosidase from Trifolium repens (orange; R91, Y326 and E397); 1PBG, phospho b-galactosidase from Lactococcus lactis (red; R72, Y299 and E374). 4872 S. R. Marana et al. (Eur. J. Biochem. 270) Ó FEBS 2003 units (Table 2). Taking into account the experimental errors, the pK a of E187 remained the same, but the pK a of E399 clearly increased. Actually, the substitution of R97 by methionine or lysine resulted in the same increment in the pK a of E399 (Table 2). Therefore, M97 and K97 are equally effective in stabilizing the charged E399. That is unexpected, because as arginine and lysine side chains are positively charged and hydrogen bond donors, they should interact similarly with E399. However, it should be considered that structural data of high resolution protein structures indicate that the geometry of the hydrogen bonds formed by those residues are very different [28] and that lysine side chain is shorter. Thus, the present results suggest that in spite of the fact that K97 could form a hydrogen bond with E399, this bond is weakened or disrupted because of unfavourable spatial positioning of interacting atoms. Alterations in the active site structure could also contribute to the observed result. In the Y331F mutant the replacement of Y331 for phenylalanine shifted the pK a of E399 by +0.7 pH units (from 4.8 to 5.5) and the pK a of E187 by +1.6 pH units (7.4–9.0). As a result, the pH optimum of the Y331F mutant (7.0) is higher than that of the wild-type Sfbgly50 (6.2) (Fig. 5). The effect of this mutation on the E399 ionization is the same as observed for the mutation R97M (DpK ES1 ¼ + 0.6; Table 2), indicating that Y331 also stabilizes the charged E 399 . Part of this effect may result from an alteration of the dielectric constant of the active site, although the deletion of the hydrogen bond between Y331 and E399 is probably a major component of that pK a increment. Based on the DpK a of E399 between the wild- type and Y331F Sfbgly50 it was calculated that the Y331 contributes 4.0 ± 0.4 kJÆmol )1 to the stabilization of the charged E399, the same value observed for R 97 .Therefore, R97 and Y331 together contribute 7.4 kJÆmol )1 to stabil- ization of the charged E399. Hence, if these two residues were removed, the ionization of E399 would be less favourable and the pK a of E399 would be higher, probably around 6.0. In the Y331F mutant, the pK a of E187 was increased by +1.6 pH units. This modification is not a result of a direct interaction between E187 and Y331 as these residues are far apart from each other. A possible explanation is that the increase in the pK a of E399 makes more difficult the ionization of E187 due to an increment in electrostatic repulsion between these glutamates. How- ever, this repulsion is not enough to completely explain the DpK a of E187 in the Y331F mutant, because in the R97K and R97M mutants the same increment in the pK a of E399 did not change significantly the pK a of E187. This suggests that in the Y331F mutant, the charged side chain of E399 may have moved to a new position closer to E187, in order to minimize unfavourable interactions with the apolar side chain of F331. Thus, the increment in the pK a of E399, in addition to it being closer to E187, may have resulted in a large shift in the pK a of E187. Changes in the dielectric constant due to F331 may further increase the pK a of E187. This unexpected shift in the pK a of E187 cannot be directly compared with data from other b-glycosidases, but the effect of the Y331F mutation in the pH-dependent activity profile is very similar to that observed in the mutant Y298F of the Agrobacterium b-glycosidase [14]. The results here obtained are similar to those found for a family 11 xylanase interaction between a tyrosine and a charged glutamate. The deletion of a hydrogen bond between a tyrosine and the catalytic nucleophile (glu- tamate)inthexylanasealsoresultedinalargeshift(+1.6 pH units) in proton donor pK a [29]. However, this comparison is not strong because family 11 does not belong to clan A [1], implying in different active site structure and composition. The mutation E187D also resulted in a large decrease in k cat for MUbglc hydrolysis (Table 1), which is explained by the aspartic acid being less efficient as a catalytic proton donor. That happens because the short D187 side chain is not as close to the glycoside bond as is the E187 side chain does and so proton donation to the leaving group (aglycone) is more difficult. In this mutant the proton donor pK a (residue 187) had a shift of )0.5 pH units (from 7.4 to 6.9) whereas, considering the errors, the nucleophile (E399) had no pK a change (Table 2). A possible explanation for this result is that there is an electrostatic repulsion between the charged E399 and E187. Thus, in the E187D mutant, this repulsion was reduced because carboxyl groups are farther apart. Consequently the proton donor ionizes more easily (pK a decreased) and the pH optimum was shifted to a value (5.8) lower than that of the wild-type Sfbgly50 (Fig. 5). Otherwise, the DpK a of the catalytic proton donor mirrors the pK a difference between free glutamic and aspartic acids (0.4 units), suggesting that an electrostatic interaction does not have any influence on the DpK a . However, the pK a values of free aspartic and glutamic acids side chains were determined in water, thus they do not have necessarily the same properties in an environment hidden from the solvent like that of the active site (less than 5% of the E187 area is exposed to the solvent). In these conditions the ionization of aspartic and glutamic acids may be equally unfavourable. Thus, if this hypothesis is correct, the DpK a of the catalytic proton donor may really indicate the presence of an electrostatic repulsion between residues E187 and E399. This hypothesis is further supported by the results obtained with b-glucosidase from Agrobacterium sp. (family 1) [4]. Here, the replacement of the catalytic nucleophile (E358) by an aspartic acid resulted in a decrease of 0.9 pH units in the pK a of the catalytic proton donor – a result also interpreted as an indication of an electrostatic repulsion between the catalytic glutamates [4]. An electrostatic repulsion between the catalytic glutamates was already described in a xylanase from family 11 [8], although one must be cautious with this comparison, as noted above. In conclusion, the combination of these results shows that residues R97 and Y331 modulate the ionization of residue E399 by stabilizing its charge and reducing its pK a , thus enabling it to function as a nucleophile. An electrostatic repulsion between ionized E399 and E187 may make E187 ionization more difficult, increasing its pK a and favouring a role as catalytic proton donor. Finally, as the pH optimum of the wild-type and mutant Sfbgly50 is an average of the E399 and E187 pK a values, it is concluded that the pH optimum of Sfbgly50 is determined by a noncovalent bonds network among R97, Y331, E399 and E187. Ó FEBS 2003 Mechanism of pH optimum control in b-glycosidases (Eur. J. Biochem. 270) 4873 Residues Y331 and R97 are totally conserved in different family 1 b-glycosidases from very different organisms (Fig. 6). The conservation of these residues suggests that, in spite of the difference in the physiological role and large evolutionary distance between the enzyme sources, those residues have the same essential function in all those b-glycosidases. Further support for this conclusion comes from a comparison between the available structures (10) of family 1 b-glycosidases. In all of these enzymes the tyrosine, arginine and glutamate (nucleophile) residues occupy a similar spatial position (in order to facilitate visualization only four are shown in Fig. 6). The distances between these residues are always in the range compatible with a hydrogen bond (3 A ˚ ), except in the Zea mays b-glycosidase, where the distance between the arginine and glutamate is 3.64 A ˚ .But even in this case, a small movement in the flexible arginine side chain would that distance shorter without any steric hindrance. This structural conservation suggests that the same noncovalent interactions are formed in all family 1 b-glycosidases. On this basis it is proposed that the noncova- lent interactions network that modulates the Sfbgly50 pH optimum is probably operating in all family 1 b-glycosidases. 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F modifications in the interactions between the catalytic glutamates and the residues R97 and Y3 31, indicating that these residues play a role in the modulation of the