Báo cáo khoa học: The role of residues R97 and Y331 in modulating the pH optimum of an insect b-glycosidase of family 1 pdf

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 frugiperdaSf

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

(pKa¼ 4.9; catalytic nucleophile) and E187 (pKa¼ 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 pKa 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 pKaof E399

and E187 by 0.7 and 1.6 units, respectively, and displaced the

pH optimum to 7.0 From the observed DpKait 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

E 187 by D decreased the pKaof 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 pKa 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; pKa 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 Accordnonreduc-ing 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)8barrel], they are

configur-ation-retaining glycosidases and their catalytic activity

depends on two glutamic acid residues, one positioned

after b strand 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 pKais around 5.0 and

the catalytic proton donor pKais around 7.0 [3–7]

A plot of b-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 pKa[8] Thus, one may hypothesize that the electrostatic coupling 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 ¼ 4.9) and E187 (catalytic proton donor, pK 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: digestive b-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)

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 pKavalues 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-GroEincreases the Gro-ELS concentration inside the cells and consequently it favours the folding of coexpressed proteins [16] The transformed bacteria were cultivated (37C, 250 r.p.m.) in Luria–Bertani broth containing carbenicillin (50 lgÆmL)1) and chloramphenicol (17 lgÆmL)1) until D600¼ 0.6–1.0 was reached The bac-teria were then induced using 1 mM isopropyl

thio-b-D-galactoside for 3 h (25C, 250 r.p.m.) and harvested at

7000 g for 20 min at 4C 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 mM Hepes buffer

pH 7.0 containing 150 mM NaCl, 0.02% (w/v) lysozyme (chicken egg white) and 0.1% (v/v) Triton X-100 The suspension was incubated at 4C 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˚.

harvested at 7000 g for 20 min at 4C 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 mM

phosphate buffer pH 7.0 containing 1.27M(NH4)2SO4, and

the retained proteins were then eluted using a gradient of

(NH4)2SO4 prepared in 50 mM phosphate buffer pH 7.0

The presence of the recombinant Sfbgly50 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 mM

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 mM

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 e280¼ 117 200

M )1Æcm)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 30C in 50 mM 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 (kcat and Km) were determined by using nine

different substrate concentrations (0.1–8 mM); enzyme

concentrations were 0.13 lM for R97M, 0.09 lM for

Y331F and 0.62 lM for E187D The data were fitted to

Michaelis–Menten equation using theENZFITTER

(Elsevier-Biosoft)

Chemical modification with phenylglyoxal

Arginine modification was performed using different

concentrations of phenylglyoxal (1, 3, 4 and 5 mM)

prepared in 20 mM phosphate buffer pH 8 at 30C In

this pH phenylglyoxal reacts specifically with arginine

residues [23,24] Wild-type (0.49 lM) or mutant Sfbgly50

(0.13 lM) samples were incubated with the modifying

agent in the absence or presence of high concentration of

NPbglc (> 4· Km) Samples were collected after

differ-ent periods of time and 10 mM arginine in 20 mM

phosphate pH 8.0 was added This material was used to

determine the remaining enzymatic activity using 4 mM

MUbglc as substrate in 50 mM citrate/phosphate buffer

pH 6.0 Then, the rate constants (kobs) for the Sfbgly50

inactivation in different phenylglyoxal concentrations were

calculated

pH effect on the Sfbgly50 activity Sfbgly50 enzymatic activity on 8 mM MUbglc was deter-mined in different buffers ranging from pH 5.0 to 8.5 (50 mMcitrate/phosphate buffer, pH 5.0–7.0; 50 mM phos-phate buffer, pH 7.0–8.0; 50 mMBicine 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 30C The enzyme concentration was 0.13 lM for mutant R97M, 0.09 lMfor mutant Y331F and 0.37 lM for mutant E187D

At 8 mMMUbglc, Sfbgly50 is approaching saturation by the substrate Hence, relative activity is a good approxima-tion of the relative maximum velocity (Vmax app) under these conditions Thus the pKas in the enzyme–substrate (ES) complex of the catalytically active groups of Sfbgly50 (pKES1and pKES2) were determined by fitting the Vmax app

of the MUbglc hydrolysis at each pH to Eqn (1) [25]

þ

KES1 þ KES2

Hþ

Vmax appis the relative Vmaxdetermined at each pH, [H+]

is the proton concentration and KES1 and KES2 are the ionization constants of the two catalytically essential groups

in the ES complex Vmax appwas expressed as a percentage

of the highest Vmaxobserved, and fitting was done using the softwareENZFITTER

Ionization constants in the free enzyme (pKE1and pKE2) were calculated using Eqn (2) [25]

kcat=Km app¼ 1

þ

KE1

þ K E2

Hþ

kcat/Km appis the relative kcat/Kmdetermined at each pH, [H+]

is the proton concentration and KE1and KE2are the ionization constants of the two catalytically essential groups in the free enzyme kcat/Km appmay be calculated from the enzymatic activity determined using low substrate concentration (0.25 mM) at different pH values and relative kcat/Km app and [H+] were fitted in the above equation usingENZFITTER The data were enough to fit simultaneously the two KES and KE In the E187D mutant, the determination of pKES1 was less accurate than that of pKES2, 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]

Sequence alignment and structural comparison

Amino acid sequences of family 1 b-glycosidases were

retrieved from the CAZy website [1] and aligned using the

softwareCLUSTALX[27] The spatial coordinates of family 1

b-glycosidases were retrieved from the PDB website and

visualized byPDBVIEWER[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 (kcatand Km) 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/PAGEof 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 , prepared in 50 m 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.

As the pKavalues of the catalytic residues determine the

pH optima, the ionization constants (pKa) of the catalytic

glutamates (E187 and E399) in the ES complex and in the

free enzyme (E) of Sfbgly50 mutants were determined The

pKESand pKEare very similar, thus only the pKESvalues are presented (Table 2) The differences observed in pKa 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 kcat, whereas Kmis 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)1Æm 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) and recombinant (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, 1 m M ; j, 3 m M ; m, 4 m M ; d, 5 m 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.

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 kcat, whereas the replacement of Y331 by F results in a 250-fold decrease (compare kcatfor the wild-type and mutant Sfbgly50 in Table 1) As the extent of kcat 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, Agrobacteriumnumbering) for a phenylalanine also results

in a large kcatdecrease (500-fold) [14]

One possible effect of R97 and Y331 on catalysis is to position E399 Thus, the kcatdecrease 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

kcat 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 pKaby +0.6 pH units (from 4.8 to 5.4), but had no effect on the E187 pKa As a consequence of the higher pKaof 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 pKa

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 ) of the

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

The observed DpKaare directly related to the differences

in the free energy change (DDG ¼ 2.303RTDpKa) 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 DpKaof 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 pKa of E399 is shifted by +0.8 pH units, whereas pK 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).

units (Table 2) Taking into account the experimental

errors, the pKaof E187 remained the same, but the pKaof

E399 clearly increased Actually, the substitution of R97 by

methionine or lysine resulted in the same increment in the

pKaof 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 pKa of E399 by +0.7 pH units

(from 4.8 to 5.5) and the pKaof 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

(DpKES1¼ + 0.6; Table 2), indicating that Y331 also

stabilizes the charged E399 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 pKa

increment Based on the DpKa of E399 between the

wild-type and Y331F Sfbgly50 it was calculated that the Y331

contributes 4.0 ± 0.4 kJÆmol)1to the stabilization of the

charged E399, the same value observed for R97 Therefore,

R97 and Y331 together contribute 7.4 kJÆmol)1to

stabil-ization of the charged E399 Hence, if these two residues

were removed, the ionization of E399 would be less

favourable and the pKaof E399 would be higher, probably

around 6.0

In the Y331F mutant, the pKa 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 pKaof 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 DpKa of E187 in the Y331F mutant, because in the

R97K and R97M mutants the same increment in the pKa

of E399 did not change significantly the pKaof 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

pKaof E399, in addition to it being closer to E187, may

have resulted in a large shift in the pKaof E187 Changes

in the dielectric constant due to F331 may further increase

the pKa of E187

This unexpected shift in the pKa 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) in the xylanase also resulted in a large shift (+1.6

pH units) in proton donor pKa [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

kcat 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 pKa (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 pKa 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 (pKa decreased) and the pH optimum was shifted to a value (5.8) lower than that of the wild-type Sfbgly50 (Fig 5) Otherwise, the DpKaof the catalytic proton donor mirrors the pKadifference between free glutamic and aspartic acids (0.4 units), suggesting that an electrostatic interaction does not have any influence on the DpKa However, the pKa 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 DpKa 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

pKaof 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 pKa, thus enabling it to function as a nucleophile An electrostatic repulsion between ionized E399 and E187 may make E187 ionization more difficult, increasing its pKaand 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 pKa values, it is concluded that the pH optimum of Sfbgly50 is determined by a noncovalent bonds network among R97, Y331, E399 and E187

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

Acknowledgements

This project is supported by FAPESP (Fundac¸a˜o de Amparo a`

Pesquisa do Estado de Sa˜o Paulo) and CNPq (Conselho Nacional de

Desenvolvimento Cientı´fico e Tecnolo´gico) E.H.P.A and L.M.F.M.

are undergraduate student fellows of CNPq S.R.M., W.R.T and C.F.

are staff members of the Biochemistry Department (IQUSP) and

research fellows of CNPq.

References

1 Coutinho, P.M & Henrissat, B (1999) Carbohydrate-active

enzymes: an integrated database approach In Recent Advances

in Carbohydrate Bioengineering (Gilbert, H.J., Davies, G.,

Henrissat, B & Svensson B., eds), pp 3–12 The Royal Society of

Chemistry, Cambridge.

2 Davies, G & Henrissat, B (1995) Structures and mechanisms of

glycosyl hydrolases Structure 3, 853–859.

3 Street, I.P., Kempton, J.B & Withers, S.G (1992) Inactivation of

a b-glucosidase through the accumulation of a stable

2-deoxy-2-fluoro-a- D -glucopyranosyl-enzyme intermediate: a detailed

investigation Biochemistry 31, 9970–9978.

4 Whiters, S.G., Ruptiz, K., Trimbur, D & Warren, R.A (1992)

Mechanistic consequence of mutation of the active site nucleophile

glu 358 in Agrobacterium b-glucosidase Biochemistry 31, 9979–

9985.

5 Keresztessy, Z., Kiss, L & Hughes, M.A (1994) Investigation of

the active site of the cyanogenic b- D -glucosidase (linamarase) from

Manihot esculenta Crantz (cassava) I Evidence for an essential

carboxylate and a reactie histidine residue in a single catalytic

center Arch Biochem Biophys 314, 142–152.

6 Wang, Q., Trimbur, D., Graham, R., Warren, R.A.J & Withers,

S.G (1995) Identification of the acid/base catalyst in

Agrobacte-rium faecalis b-glucosidase by kinetic analysis of mutants.

Biochemistry 34, 14554–14562.

7 Skoubas, A & Georgatsos, J.G (1997) Identification of essential

amino acids for the catatytic activity of barley b-glucosidase.

Phytochemistry 46, 997–1003.

8 McIntosh, L.P., Hand, G., Johnson, P.E., Joshi, M.D., Ko¨rner,

M., Plesniak, L.A., Ziser, L., Wakarchuk, W.W & Withers, S.G.

(1996) The pK a of the general acid/base carboxyl group of a gly-cosidase cycles during catalysis: a13C-NMR study of Bacillus circulans xylanase Biochemistry 35, 9958–9966.

9 Marana, S.R., Jacobs-Lorena, M., Terra, W.R & Ferreira, C (2001) Amino acid residues involved in substrate binding and catalysis in an insect digestive b-glycosidase Biochim Biophys Acta 1545, 41–52.

10 Burmeister, W.P., Cottaz, S., Driguez, H., Iori, R., Palmieri, S & Henrissat, B (1997) The crystal structures of Sinapis alba myr-osinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of

an S-glycosidase Structure 15, 663–675.

11 Barrett, T., Suresh, C.G., Tolley, S.P., Dodson, E J & Hughes, M.A (1995) The crystal structure of a cyanogenic b-glucosidase from white clover, a family 1 glycosyl hydrolase Structure 3, 951–960.

12 Sanz-Aparicio, J., Hermoso, J.A., Martı´nez-Ripoll, M., Leq-uerica, J.L & Polaina, J (1998) Cristal structure of b-glucosidase

A from Bacillus polymyxa: Insights into the catalytic activity in family 1 glycosyl hydrolases J Mol Biol 275, 491–502.

13 Aguilar, C.F., Sanderson, I., Moracci, M., Ciaramella, M., Nucci, R., Rossi, M & Pearl, L.H (1997) Crystal structure of the b-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus: Resilence as a key factor in thermostability J Mol Biol 271, 789–802.

14 Gebler, J.C., Trimbur, D.E., Warren, A.J., Aebersold, R., Nam-chuk, M & Withers, S.G (1995) Substrate-induced inactivation of

a crippled b-glucosidase mutant: identification of the labeled amino acid and mutagenic analysis of its role Biochemistry 34, 14547–14533.

15 Tabor, S & Richardson, C.C (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression

of specific genes Proc Natl Acad Sci USA 82, 1074–1078.

16 Takashi, Y., Kanei-Ishii, C., Maekava, T., Fujimoto, J., Yama-moto, T & Ishii, S (1995) Increase of solubility of foregein pro-teins in Escherichia coli by co-production of the bacterial thioredoxin J Biol Chem 270, 25328–25331.

17 Laemmli, U.K (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685.

18 Terra, W.R., Ferreira, C & Bianchi, A.G (1979) Distribution of digestive enzymes among the endo and ectoperithophic spaces and midgut cells of Rhynchosciara americana and its physiological significance J Insect Physiol 25, 487–494.

19 Blum, H., Beir, H & Gross, H (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels Electro-phoresis 8, 93–99.

20 Gill, S.C & von Hippel, P.H (1989) Calculation of proteins extinction coefficients from amino acid sequence data Anal Biochem 182, 319–326.

21 Marana, S.R., Terra, W.R & Ferreira, C (2000) Purification and properties of a b-glycosidase purified from midgut cells of Spodoptera frugiperda (Lepidoptera) larvae Insect Biochem Mol Biol 30, 1139–1146.

22 Baker, J.E & Woo, S.M (1992) b-glucosidases in the rice weevil, Sitophilus oryzae: purification, properties, and activity levels in wheat-and legume feeding strains Insect Biochem Mol Biol 22, 495–504.

23 Takahashi, K (1968) The reaction of phenylglyoxal with arginine residues in proteins J Biol Chem 243, 6171–6179.

24 Yamasaki, R.B., Vega, A & Feeney, R.E (1980) Modification of available arginine residues in proteins by p-hydroxyphenylglyoxal Anal Biochem 109, 32–40.

25 Segel, H.I (1993) Enzyme Kinetics Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems John Wiley & Sons, New York.

26 Guex, N & Peitsch, M.C (1997) SWISS-MODEL and the

Swiss-PdbViewer: An environment for comparative protein modelling.

Electrophoresis 18, 2714–2723.

27 Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F &

Higgins, D.G (1997) The CLUSTAL_X windows interface:

flex-ible strategies for multiple sequence alignment aided by quality

analysis tools Nucleic Acids Res 25, 4876–4882.

28 Ippolito, J.A., Alexander, R.S & Christianson, D.W (1990) Hydrogen bond stereochemistry in protein structure and function.

J Mol Biol 215, 457–471.

29 Joshi, M.D., Sidhu, G., Nielsen, J.E., Brayer, G.D., Withers, S.G.

& McIntosh, L.P (2001) Dissecting the eletrostatic interactions and pH-dependent activity of a family 11 glycosidase Biochem-istry 40, 10115–10139.