172

Biochimica et Biophysica Acta, 1041 (1990) 172 177

Elsevier BBAPRO 33763

Chemical modification of xylanases: evidence for essential tryptophan and cysteine residues at the active site * Vasanti Deshpande, Jyoti Hinge and Mala Rao Division of Biochemical Sciences, National Chemical Laboratory, Pune (India)

(Received 12 January 1990) (Revised manuscript received20 June 1990) Key words: Xylanase;Tryptophan; Cysteine;Chemicalmodification

N-Bromosuccinimide (NBS) completely inactivated xylanases from Chainia and alkalophilic and thermophilic (AT) Bac///us with a concomittant decrease in absorption at 280 nm and with second-order rate constants of 1 0 ~ 0 and 5000 M - 1 . min-l, respectively at pH 6.0 and 25 ° C. The kinetic analysis of inactivation indicated that one and three tryptophan residues were essential for the xylanase activity from Cha/n/a and Bac/Uus, respectively. The xylanases were also inhibited by 2-hydroxy-5-nitrohenzyl bromide (HNBB). The modification of cysteine residues by p-hydroxymercuryhenzoate (PHMB) and N-ethylmaleimide did not cause a loss in activity of the xylanase from Bac//&s, whereas that from Cka/n/a was completely inactivated. The kinetics of inactivation revealed the involvement of one cysteine residue for xylanase from Cha/n/a with a second-order rate constant of 50000 M - I . min -1. The PHMB-modifled enzyme failed to show the presence of titrable -SH groups. Xylan afforded complete protection against inactivation by NBS, HNBB and PHMB, indicating the involvement of tryptophan and cysteine residues at the substrate-binding region of the enzyme. Introduction Xylanases (1,4-fl-D-xylan xylanohydrolase; EC 3.2.1.8) catalyze the hydrolysis of xylan to xylooligosaccharides and xylose which are useful feed stock for generating food and fuel [1]. Xylan is the major component of hemicellulose which represents one third of polysaccharide contents of the renewable plant biomass [2]. Xylanases have a potential application in the paper and pulp industry for selective hydrolysis of hemicelluloses [3]. The production, purification and synergism of the components of xylanase have been investigated [4-6]. In spite of the biotechnological importance, only limited information concerning the mechanism of action of these enymes is available. In contrast, the catalytic mechanism of lysozyme and cellulases which are functionally related to the xylanases, have been the subject of intensive investigations [7,8]. The biological importance of xylanases and their potential relationship to lysozyme and cellulase make them excellent candi* NCL CommunicationNo. 4627. Abbreviations: NBS, N-bromosuccinimide; PHMB, p-hydroxymereurybenzoate; DTN'B, 5,5-dithiobis(2-nitrobenzoate); HNBB, 2hydroxy-5-nitrobenzylbromide. Correspondence: M. Rao, Divisionof BiochemicalSciences,National Chemical Laboratory,Pune 411 008, India.

dates for active site studies. Investigations involving chemical modification of an enzyme can potentially yield insights into structure-function relationships. Studies on the mode of action of endoxylanases have been carried out by subsite mapping using labelled xylooligosaccharides [9,10]. Few reports on the involvement of tryptophan and cysteine residues in the active site of xylanases from bacterial systems are known, but detailed studies have not been carried out [11-13]. The xylanases from alkalophilic organisms are stable and active at alkaline pH. Also low molecular weight xylanases have greater access to the cellulose pulp. In the present paper, the amino acid residues involved in the active site of a xylanase ( M r 35000) from an alkalophilic and thermophilic Bacillus [14] and of a low molecular weight xylanase ( M r 6000) from Chainia [13] have been investigated. Kinetic and chemical modification studies show the presence of tryptophan residues at the substrate-binding site of both the xylanases. Evidence is also presented for the involvement of a cysteine residue at the active site of xylanase from Chainia.

Materials and Methods Materials

N-Bromosuccinimide (NBS), N-ethylmaleimide, diethylpyrocarbonate, p-hydroxymercurybenzoate (PH-

0167-4838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (BiomedicalDivision)

173 MB), phenyl glyoxal, iodoacetamide, N-acetylirnidazole 3,5-dinitro-salicylic acid, phenylmethylsulfonyl fluoride 5,5-dithiobis(2-nitrobenzoate) (DTNB) and 2-hydroxy5-nitrobenzyl bromide (HNBB) were purchased from Sigma Chemical Co. St. Louis, MO, U.S.A. Xylan (larch wood) and cysteine were from Fluka AG, Switzerland. All the other chemicals were of analytical grade.

(94-100). Xylanase I fractions were pooled, dialyzed against water and concentrated by lyophilization and were used for the present studies. The xylanase from Chainia was purified by chromatography on DEAE-cellulose, and by gel filtration on Sephadex G-50 [13]. SDS gel electrophoresis was performed according to Laemmli [19].

Microorganisms The alkalophilic and thermophilic (AT) Bacillus sp. was isolated from Vajreshwari in Maharashtra, India. A wheat bran (5%) yeast extract (1%) medium at pH 10 at 50°C was used for the isolation. The organism was further purified by single colony plating techniques. Chainia sp. was isolated from a soil sample collected near Haldighat, Rajastan [15].

Titration with DTNB The free sulfhydryl groups in the enzyme were determined by DTNB titration [20]. The enzyme (2.5 × 10-5 M) in 1 ml of 0.05 M potassium phosphate buffer (pH 7:5) containing 1% sodium dodecyl sulphate was treated with 0.02 ml of 0.1 M DTNB. Increase in absorbance at 412 nm was measured. In a separate experiment the enzyme was treated with 10 -4 M PHMB prior to DTNB titration.

Enzyme assay Xylanase was assayed by mixing 0.5 ml of xylan solution (1%) incubating at 50°C for 30 min. The reducing sugar released was determined by Miller's method [16] using D-xylose as standard. Two g of xylan were suspended in 100 ml of 50 mM sodium acetate buffer (pH 5.7) and stirred for 12 to 16 h. The insoluble fraction was removed by centrifugation and the soluble fraction was used for xylanase assay. The unit of xylanase was defined as that amount of enzyme which produced 1 /~mol of xylose equivalent per min from xylan under the assay conditions. The protein concentration was measured according to Lowry et al. [17]. Enzyme production Enzyme was produced in 250 ml flasks with 50 ml medium containing wheat bran (5%) and yeast extract (1%). The vegetative inoculum (10%) was grown in the same medium except for containing 1% wheat bran for 18 h. The pH of the medium for AT Bacillus was adjusted to 10 with 1% Na2CO 3 and was grown for 48 h at 48-50°C. Chainia was grown for 76 h at 28°C at pH 7 [18].

Purification The culture filtrate of AT Bacillus (400 ml) was concentrated by precipitating the enzyme with 3 vol. of chilled ethanol. The alcohol precipitated culture filtrate was recovered by centrifugation and dried under vacuum. The precipitate was dissolved in 10 ml of 25 mM sodium acetate buffer (pH 6). 2 ml (1600 U) of the alcohol precipitated enzyme broth was applied to BioGel P-10 column (4 X 110 cm) equilibrated with 25 mM sodium acetate buffer (pH 6.0). The fractions (3 ml each) were collected and estimated for xylanase activity. The eluates showed two peaks of xylanase activity. Xylanase I fraction (32-40) and Xylanase II fraction

Reaction of chemical modifiers with xylanase The effect of a number of chemical inhibitors on xylanase activity was tested by incubating 5.0 #g of enzyme at 25 °C with varying concentrations of modifier in a total of 2.5 ml of appropriate buffer. Aliquots of 0.5 ml were removed periodically for the measurement of residual enzyme activity. Control tubes having only enzyme or only inhibitor or inhibitor and substrate were incubated under identical conditions. The pseudo-first-order rate constants (K) were obtained from the slopes of the plots of logarithm of the residual activity against time of reaction. The secondorder rate constants were calculated from the slopes of the plots of pseudo-first-order rate constants against concentration of inhibitor [21]. Titration with NBS Oxidation of tryptophan residues by NBS was carried out in two cuvettes. One containing xylanase in 50 mM acetate buffer (pH 5) and the other containing only buffer. Successive 10/~l aliquots of NBS were added to the sample as well as to the reference cuvette and absorbance at 280 nm was measured. The xylanase from Chainia (6.7.10 -s M) was titrated against 1.10 -2 M N BS and that from AT Bacillus (3 • 10-6 M) was titrated against 1 • 1 0 - 4 M NBS. Aliquots of the reaction mixture were assayed for xylanase activity simultaneously. Results

The purified xylanase from the AT Bacillus (Table I) showed a single band on SDS-gel electrophoresis and had an M r of 35 000. It has a wide pH optimum in the range of 6 to 9 (manuscript under preparation). The molecular weight of xylanase from Chainia was determined to be 5000-6000 by gel filtration, SDS-gel electrophoresis, amino acid composition and by amino acid sequencing [13]. It had optimum activity at pH 6.0.

174 TABLE I Purification of xylanase I from A T Bacillus

Fraction

Total volume (ml)

Total Total SPA protein activity U/mg (mg) (units)

Culture filtrate Ethanol precipitation Biogel chromatography

400 10 1

800 250 0.1

12000 5000 10

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15 20 100

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The importance of particular functional groups for the activity of the xylanases was investigated by the use of reagents with restricted amino acid specificity. Diethyl pyrocarbonate, N-acetyl-imidazole, phenylglyoxal and phenylmethylsulfonyl fluoride were not inhibitory to xylanases from AT Bacillus and Chainia indicating that histidine, tyrosine, arginine and serine are not involved in the active site (Table II). NBS and HNBB were potent inhibitors of both the xylanases suggesting a crucial role of tryptophan residues. PHMB and N-ethylmaleimide inhibited the xylanase activity from Chainia but had no effect on the xylanase from A T Bacillus. Based on these results, the involvement of tryptophan and cysteine residues in xylanase activity was further investigated.

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Fig. 1. Effect of NBS on xylanase from AT-Bacillus. Enzyme (5 #g) was incubated with NBS: 0.05 mM (e). 0.055 mM (o), 0.06 mM (zx), and 0.07 mM (121).Inset: Apparent order of reaction with respect to the reagent concentration. The observed pseudo-order rate constants calculated from Fig. 1 were plotted.

activity against the time of reaction. A double-logarithmic plot of the observed pseudo-first-order rate constant against reagent concentration yielded a reaction order of 3.0 for A T Bacillus xylanase and 1.0 for Chainia xylanase indicating that the modification of three and one residues results in the loss of enzyme activity (Figs. 1 and 2, insets). The second-order rate constants for inactivation of xylanases from Chainia and A T Bacillus were calculated to be 10 500 and 5000 M - 1. m i n - 1, respectively. The titration curves of xylanase from AT Bacillus and Chainia with NBS are shown in Fig. 3a and b,

Reaction o f xylanase with N-bromosuccinimide Incubation of NBS with xylan showed an optical density equivalent to that of xylan alone indicating that NBS does not react with xylan. Treatment of xylanases with NBS led to inactivation which was dependent both upon the time and reagent concentration. Plots of the log of residual activity versus time at all concentrations of the reagent were linear, indicating that the inactivation follows first order kinetics (Figs. 1 and 2). Applying the analysis described by Levy et al. [21] the pseudo-first-order rate constants were calculated from the slope of the plots of logarithm of the residual

TABLE II Effect of chemical inhibitors on xylanase activity

The experimental details are described in Materials and Methods Chemical

Inhibitor concentration in reaction mixture (raM)

Residual activity AT-Bacillus Chamia (~) (~)

Incubation buffer (50 mM)

Diethyl pyrocarbonate N-Acetylimidazole Phenylglyoxal Phenylmethylsulfonylfluoride Iodoacetamide N-Ethylmaleimide p- Hydroxymercurybenzoate N-Bromosuccinimide 2-Hydroxy-5-nitrobenzylbromide

10 10 10 10 10 10 1 1 10

100 100 100 100 100 100 100 0 20

potassium phosphate buffer (pH 6.0) potassium phosphate buffer (pH 7.0) Tris-HC1 buffer (pH 8.0) potassium phosphate buffer (pH 7.0) sodium acetate buffer (pH 6.0) sodium a~tate buffer (pH 6:0) sodium acetate buffer (pH 6.0) sodium acetate buffer (pH 6.0) sodium acetate buffer (pH 6.0)

100 100 100 100 70 60 0 0 30

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Fig. 2. Kinetics of inactivation of xylanase from Chainia by NBS. Xylanase (5 /~g) was incubated with NBS 1.5 ~M (e), 3.5 /LM (X), and 6/~M (o). Inset: Apparent order of reaction with NBS.

Fig. 4. Influence of PHMB on the inactivation of xylanase from Chainia. Enzyme (5 #g) was incubated with PHMB 0 #M (o), 0.5 #M (e), 0.75 ~M (rn) and 1 ~M (zx). Inset: Apparent order of reaction with PHMB.

respectively. A progressive decrease in absorption at 280 nm was observed after each addition of NBS. on the rate of inactivation of xylanase from Chainia is shown in Fig. 4. Plots of percent residual activity as a function of time at various concentrations indicate that the inactivation process exhibits pseudo-first-order kinetics with respect to time at any fixed concentration of the inhibitor. The order of the reaction was estimated to be 1.0 from the slopes of the plots of logarithm of pseudo-first-order rate constant vs. log of inhibitor concentration (Fig. 3, inset). The second-order rate constant was calculated to be 50000 M -~. min ~ . Treatment with cysteine of PHMB-inactivated xylanase resulted in 82% reactivation of the enzyme, indicating that inhibition is due to the modification of cysteine residue.

Titration of chainia xylanase with DTNB Treatment of the xylanase from Chainia with D T N B for 1 h showed an increase in absorbance of 0.250 which corresponds to 0,83 mol of cysteine per tool of enzyme assuming a molecular weight of 6000. However, when the enzyme was treated with PHMB prior to D T N B there was no increase in absorbance, indicating that the -SH group wa~ not available for titration with DTNB. Reaction of Chainia xylanase with PHMB A variety of thiol reagents inactivated the xylanase at widely different rates (Table II). The effect of PHMB

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Chemical modification of xylanases: evidence for essential tryptophan and cysteine residues at the active site.

N-Bromosuccinimide (NBS) completely inactivated xylanases from Chainia and alkalophilic and thermophilic (AT) Bacillus with a concomittant decrease in...
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