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Chemical modification of xylanase from alkalothermophilic Bacillus species: evidence for essential carboxyl group
BB
ELSEVIER
Biochimica et Biophysica Acta 1204 (1994) 164-168
Biochi~ic~a et Biophysicat~ta
Chemical modification of xylanase from alkalothermophilic Bacillus species: evidence for essential carboxyl group 1 Jyoti Chauthaiwale, Mala Rao
*
Division of Biochemical Sciences, National Chemical Laboratory, Pune - 411008, India
(Received 29 April 1993; revised manuscript received 7 September 1993)
Abstract
The role of carboxyl group in the catalytic action of xylanase (M r 35 000) from an alkalothermophilic Bacillus sp. was delineated through kinetic and chemical modification studies using Woodward's Reagent K. The kinetics of inactivation indicated that one carboxyl residue was essential for the xylanase activity with a second order rate constant of 3300 M - 1 min- l. The spectrophotometric analysis at 340 nm revealed that the inhibition was correlated with modification of 24 carboxyl residues. In the presence of protecting ligand, modification of one carboxyl group was prevented. The pH profile showed apparent pK values of 5.2 and 6.4 for the free enzyme and 4.9 and 6.9 for enzyme-substrate complex. The pH dependence of inactivation was consistent with the modification of carboxyl group. The kinetic analysis of the modified enzyme showed similar K m and lower kca t values than the native enzyme indicating that catalytic hydrolysis and not the substrate binding was affected by chemical modification. The chemical modification of xylanase from alkalothermophilic Bacillus revealed the presence of tryptophans in the active site (Deshpande, V, Hinge, J. and Rao, M. (1990) Biochim. Biophys. Acta 1041, 172-177). This finding and present studies demonstrated the experimental evidence for the participation of carboxyl as well as tryptophan groups as essential residues of xylanase from alkalothermophilic Bacillus sp. Key words: Chemical modification; Xylanase; Alkalothermophilicity; Essential carboxyl group; (Bacillus sp.)
1. Introduction
Xylanase (1,4-/3-D-xylanohydrolase, EC 3.2.1.8) catalyzes the random hydrolysis of xylan to xylooligosaccharides and xylose which are useful feed stock for generating food and fuel [1]. Xylanases in conjunction with cellulases are useful for the complete conversion of cellulosic biomass to sugars [2]. The use of cellulase-free xylanases for the selective hydrolysis of the hemicellulose components in p a p e r and pulp has also received considerable attention [3]. Investigations on the roles of critical amino-acid residues of an enzyme are important in structure function analysis and protein engineering studies. The chemical modification and kinetic analysis of carbohydrases such as amylases [4,5] and cellulases [6,7] have revealed that tryptophan and carboxyl groups are involved in the catalytic activ-
* Corresponding author. Fax:+ 91212 334761. 1 NCL Communication No. 5766. 0167-4838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4838(94)E0189-9
ity. The involvement of carboxyl groups for the catalysis [8] and the participation of tryptophan residues for the substrate binding [9] have been shown for lysozyme. The studies on sequence similarities of xylanase, cellulase and lysozyme have supported the possibility that catalytic reaction of these enzymes follows the same mechanistic pathway [10]. Inspite of tremendous biotechnological applications of xylanases, little information is available on the mechanism of catalysis. However, there is one report on chemical modification of xylanase from fungus SchizophyUum c o m m u n e indicating the involvement of carboxyl groups in the catalysis [11]. Site-directed mutagenesis of xylanase from Bacillus p u m i l u s has shown that Glu-93 and Glu-182 are the essential catalytic residues [12]. The participation of tryptophan and cysteine residues at the active site of xylanases from Chainia [13] and Streptomyces T 7 [14] has been reported. The chemical modification studies showed the involvement of tryptophan residues at the catalytic site [13] of the xylanase (M r 35000) from alkalothermophilic Bacillus sp. [15,16]. The analysis of
J. Chauthaiwale,M. Rao / Biochimica et BiophysicaActa 1204 (1994) 164-168
the competitive inhibition of the xylanase (M r 35 000) by guanidine hydrochloride has shown the specific interaction between 'arginine like' guanidine moiety and carboxyl group at the active site of the enzyme [17]. The present paper describes the kinetic analysis and chemical modification studies that provide experimental evidence for the participation of essential carboxyl group in the catalytic mechanism of the xylanase of alkalothermophilic Bacillus. Our earlier findings [13] and present studies demonstrated the evidence for the presence of carboxyl as well as tryptophan groups as the essential residues of xylanase from alkalothermophilic Bacillus.
2. Materials and methods
2.1. Materials
Oat spelt xylan, standard amino-acid mixture, Woodward's Reagent K (2-ethyl-5-phenyl isoxazolium3-sulfonate) [WRK], dithionitrobenzoic acid (DTNB) and ethylamine were purchased from Sigma (USA). All other chemicals were of analytical grade. 2.2. Production and purification of enzyme
Alkalothermophilic Bacillus was grown in 250 ml flasks with 50 ml medium containing wheat bran (10%), yeast extract (1%) and Na2CO 3 (1%). The culture was incubated at 50°C and pH 10.0 for 48 h. The enzyme was purified by alcohol precipitation, gel filtration and preparative polyacrylamide gel electrophoresis as described earlier [15]. 2.3. Enzyme assay
The suitably diluted enzyme in 50 mM potassium phosphate buffer (pH 7.0) was incubated with substrate 1% xylan in a final volume of 1.0 ml at 50°C for 30 min. The reducing sugar released was determined by Miller's method [18] using D-xylose as standard. The unit of xylanase was defined as the 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 Bradford et al [19]. 2.4. Amino-acid composition
The amino-acid composition of the xylanase was performed in Spinco model 120-B automatic amino-acid analyzer by the method of Spackman et al. [20]. Protein (1 mg) was hydrolyzed for 24 h, 48 h and 72 h at ll0°C with 6 M HC1 in tubes that had been sealed under vacuum. Total cysteine content was determined by
165
performic acid oxidation [21]. The tryptophan and tyrosine contents were determined by the method of Goodwin and Morton [22]. Reduction of disulfide linkages with NaBH 4 in 8 M urea and subsequent titration with DTNB were performed [23]. 2.5. Modification by WRK
Xylanase (15 /xg) in 50 mM potassium phosphate buffer (pH 6.0) was incubated with different concentrations of WRK (5-30 mM) at 25°C. Aliquots of reaction mixture were withdrawn at indicated time intervals and excess reagent was quenched with sodium acetate buffer (pH 5.0) (final concentration 250 mM). The residual xylanase activity was measured and expressed as percentage of a control. Control tubes having enzyme or inhibitor with and without substrate were incubated under identical conditions. The pseudo-firstorder rate constants were obtained from the slopes of the plots of logarithm of the residual activity against time. The second order rate constant was calculated from the slope of the plot of pseudo-first-order rate constants against concentration of inhibitor [24]. Xylanase was incubated with different concentrations of WRK (1-60 mM) for 30 min, the reaction was quenched with sodium acetate buffer (pH 5.0) and the samples were passed through Bio-Gel P-10 column equilibriated and eluted (1 m l / 2 min) with 50 mM potassium phosphate buffer (pH 6.0). Aliquots were removed to estimate the residual activity. The degree of modification was measured by the increase in absorbance at 340 nm (7000 M -1 cm -1) [25]. The modification of xylanase in presence and absence of xylose (20 mM) was performed similarly with the estimation of residual activity. The enzyme modified with WRK (50 mM, 30 min) was hydrolyzed for 24 h and analyzed for amino-acid residues as described earlier in this section. Ethylamine and standard amino-acid mixture were used as standards.
3. Results and discussion
3.1. Amino-acid analysis
Amino-acid analysis of the xylanase from alkalothermophilic Bacillus showed predominance of acidic amino acids and glycine (Table 1). Spectrophotometric analysis of protein sample by Goodwin and Morton method indicated the presence of 10 tryptophan residues per mole of enzyme and the ratio of tryptophan to tyrosine was found to be 5:1. Analysis of sample oxidized with performic acid showed the presence of one cysteine residue per enzyme molecule. Presence of one SH group was also indicated by the
J. Chauthaiwale, M. Rao / Biochimica et Biophysica Acta 1204 (1994) 164-168
166
Table 1 Amino-acid composition of xylanase from alkalothermophilic Bacillus Amino acid
Gly Ala Thr Ser Pro Asx Glx His Lys Arg Val Ile Leu Met Trp Tyr Phe Cys
Composition (residues/mol of enzyme)
mole%
98 4 2 2 1 66 158 1 2 1 1 2 2 3 10 2 1 1
27.88 + 0.10 1.00 + 0.08 0.44 + 0.10 0.46 + 0.06 0.17 + 0.03 18.98 + 0.10 45.00 + 0.12 0.18 + 0.02 0.51 + 0.04 0.21 + 0.03 0.23 + 0.07 0.43 + 0.06 0.44 + 0.04 0.76 + 0.10 2.72 + 0.20 0.43 + 0.01 0.15 + 0.01 0.23 + 0.01
DTNB titration of denatured xylanase. Analysis of reduced enzyme indicated the absence of disulfide bridge.
3.2. Chemical modification of xylanase by WRK The reaction of the enzyme with the carboxyl group specific Woodward's Reagent K resulted in progressive loss of activity. When enzyme was incubated with 20 mM WRK for 30 rain the extent of inactivation observed was 90%. Inactivation of xylanase by WRK was dependent upon the time and reagent concentration but the pattern of inhibition was complex as shown by the biphasic nature of semilogarithmic plots of residual activity as a function of time (Fig. 1). The course of inhibition can be resolved into two first order processes with the slope at longer times determining the rate of inactivation. The biphasic nature of the kinetics of inactivation can be explained on the basis of apparently large number of carboxyl residues exposed to the action of the WRK. The initial inactivation of the enzyme might be due to the modification of readily available non-catalytic carboxyl groups which might cause slight alteration in the microenvironment of the active site. During the second phase the modifier specifically reacts with the carboxyl groups located at the active site causing time- and concentration-dependent inhibition. The biphasic nature of inactivation is a common phenomenon among the carboxyl group modification of a variety of enzymes including carbohydrases [26,27]. A double-logarithmic plot of the observed pseudo-first-order rate constants against reagent concentration yielded an order of 1.0 indicating that
V- i.s
it ~c 0 . 8
~
1.6 J
I i.o LOG
I 1.5
[WRK]mM
0 .J I-2
I io
I 20 TIME
l 30
I 4O
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Fig. 1. Kinetics of inactivation of xylanase from alkalothermophilic Bacillus: Xylanase (15/xg) was incubated with WRK 5 mM (o), 10 mM ( x ) , 20 mM (zx) and 30 mM ([]) W R K at 25°C. Aliquots of reaction mixture were removed at indicated time-intervals. The residual xylanase activity was measured and expressed as percentage of control. Inset: Apparent order of reaction with respect to reagent concentration: The pseudo-first-order rate constants (k) were calculated from the slopes of semilogarithmic plots of residual activity against time.
modification of a single carboxyl residue resulted in the loss of enzyme activity (Fig. 1 inset). The second order rate constant was calculated to be 3300 M-1 min-1. The reaction of WRK is initiated by the formation of ketoketenimine which modifies the carboxyl group of the enzyme to give an enol ester with concomitant increase in the absorbance at 340 nm [25]. The spectral analysis of WRK modified xylanase at 340 nm revealed the modification of 24 carboxyl residues per molecule of the enzyme. However in the presence of xylose, the competitive inhibitor of the xylanase, 23 carboxyl residues per molecule of the enzyme were modified indicating the presence of a single carboxyl group at the active site (Table 2). The possibility of interference of sulfhydryl groups in the spectral analysis can be excluded, since DTNB titration of native xylanase did not show any change in the absorbance indicating the absorbance at 340 nm was entirely owing to carboxyl group modification. Fig. 2 shows the residual enzymatic activity plotted against the number of carboxyl groups modified. The Table 2 Differential modification of xylanase by WRK in presence and absence of xylose, a competitive inhibitor No.
Sample
Residual activity (%)
Reading at 340 nm
No. of carboxyl groups modified
1 2 3
Enzyme Enzyme + WRK Enzyme + xyiose + W R K
100 20 80
0.018 0.523 0.506
24.04 23.20
Xylanase (100 /zg) was modified with WRk (50 mM, 30 min) in presence or absence of 20 mM xylose.
J. Chauthaiwalei M. Rao / Biochirnica et Biophysica Acta 1204 (1994) 164-168
167
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,
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,
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Fig. 3. Determination of K m and kcat for native xylanase ( × ) and xylanase mofidied by WRK (<3). The native and partially modified (10 mM WRK for 10 min) enzymes were allowed to react with different concentrations of xylan (1-10 mg) and the activity was estimated. The values for K m and Kcat were determined from Lineweaver-Burk plot.
t.O IM reI
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NO. OF CARBOXYL GROUPS MODIFIED
Fig. 2. Plot of percentage residual activity against number of carboxyl groups modified. Xylanase (100 tzg) was incubated with different concentrations of WRK (1-60 mM) for 30 min. The residual activity was determined and the samples were analyzed spectrophotometrically at 340 nm. The number of carboxyl groups modified was determined by assuming molar absorption coefficient of 7000 M-1 cm-
1.
extrapolation with the Y- intercept passing through 0% inactivation showed the presence of one carboxyl group at the catalytic site. A comparison of the amino-acid composition of the unmodified and WRK modified xylanase revealed that contents of amino-acid residues other than Asx and Glx were not altered (data not shown).The modification of carboxyl residues was confirmed by the liberation of ethylamine [25].
5.2 and 6.4 for the free enzyme and 4.9 and 6.9 for the enzyme-substrate complex suggesting the protonated forms of aspartic a n d / o r glutamic acid. The pH oriented inactivation and the absence of histidyl group at the active site [13] indicated the ionizable groups as carboxylates. The pK value of the xylanase towards neutrality can be ascribed to a carboxyl group. The higher pK values of glutamic acid, as observed in case of lysozyme, is attributed to the fact that the residue is located in a non-polar environment [28]. The presence of tryptophan at the active site may create a hydrophobic environment. Ionization of a neighboring acidic residue has been shown to increase the pK value. The binding of substrate, especially long-chain substrate, induces local conformational changes that alters the microenviron-
3.3. Kinetic analysis: pH dependence The kinetic parameters Michaelis-Menten constant (K m) and turnover number ( k c a t) w e r e obtained from Lineweaver-Burk plots of the data (Fig. 3). The K m of WRK modified enzyme was found to be comparable (1.81 mg) to that of native enzyme (1.58 mg) at pH 6.0. However, the catalytic rate constant of modified enzyme was calculated to be 2.13.10 -3 S -1 which was three orders of magnitude lower than the native xylanase (6.17.10 -3 S -1) indicating that the catalytic hydrolysis and not the substrate binding is affected by the chemical modification. The inactivation of xylanase by WRK was found to be pH dependent and maximum inhibition occurred at pH 6.0 (Fig. 4). The plot of log K against pH showed a bell-shaped graph indicating the presence of two ionizable groups with pK values 5.3' and 6.4. Semilogarithmic plots of kcat and kcat.Km 1 against pH are shown in Fig. 5. These profiles indicated apparent pK values of
IO o_ 0 . 8 X v ~d ~9
\
0"6
o
_i
I 4
i 5
I 6
~ 7
I 8
pH Fig. 4. pH-dependent WRK modification of xylanase: Xylanase (15 /.~g) was modified by WRK (10 mM) in 50 mM buffers of different pH (pH 4 and 5, KCI/HCI; pH 6-8 potassium phosphate). Aliquots were removed after indicated time-intervals and were checked for xylanase acitivity. The pseudo-first-order rate constants were obtained from the slopes of the plots of logarithm of residual activity against time.
168
J. Chauthaiwale, M. Rao / Biochimica et Biophysica Acta 1204 (1994) 164-168 ,¢
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References
1.4
1.2 1.0 o --
ship a w a r d e d by t h e University G r a n t s C o m m i s s i o n to Jyoti V. C h a u t h a i w a l e a r e gratefully a c k n o w l e d g e d .
0-8 0.6
v
o 0.4 o Jig --, 0 . 2
0 _1
I 4
I 5
I 6
I 7
i 8
pH
Fig. 5. Dependence of kinetic parameters on pH: Xylanase (3 /zg) was reacted with different concentrations of substrate (1-10 mg of soluble xylan) in 50 mM buffers of pH ranging from 4-8. K m and Kca t values were derived from Lineweaver Burk plots. Effect of pH on (a) log kca t (b) log kcat.Km1.
m e n t o f acidic a m i n o - a c i d r e s i d u e resulting in an inc r e a s e in its p K v a l u e [29]. I n case o f lysozyme t h e high p K value o f Glu-35 is c o n s i d e r e d to b e critical for the c a r b o x y l a t e g r o u p to act at h i g h e r p H [28]. T h e xylanase f r o m a l k a l o t h e r m o p h i l i c Bacillus s h o w e d optim u m activity at p H 6.0 b u t was also active at h i g h e r p H [15] which m a y b e d u e to t h e h i g h e r p K value o f e s s e n t i a l carboxyl group. T h e p r e s e n c e o f carboxyl r e s i d u e at t h e active site of xylanase as i n d i c a t e d in t h e p r e s e n t studies is consist e n t with t h e o b s e r v a t i o n t h a t a s p a r t i c acid r e s i d u e involved in t h e catalysis is c o n s e r v e d a m o n g m i c r o b i a l g l y c o h y d r o l a s e s [30].
Acknowledgments
T h e p a r t i a l s u p p o r t by t h e D e p a r t m e n t o f Biotechnology ( G o v e r n m e n t o f I n d i a ) a n d t h e r e s e a r c h fellow-
[1] Jeffries, T.W. (1981) Biotechnol. Lett. 3, 213-218. [2] Biely, P. (1985) Trends Biotechnol. 3, 286-290. [3] Jurasek, L. and Paice, M. (1986) Chem. Tech. (Heidelberg) 16, 360-365. [4] Kochhar, S. and Dua, R.D. (1984) Biosci. Rep. 4, 613-619. [5] Kochhar, S. and Dua, R.D. (1985) Biochem. Biophys. Res. Commun. 126, 973-996. [6] Hurst, P.L., Sullivan, P.A. and Shephard, M.G. (1977) Biochem. J. 167, 549-556. [7] Clark, A.J. and Yaguchi, M. (1985) Eur. J. Biochem. 149, 223-238. [8] Vernon, C.A. (1967) Proc. R. Soc. London. B 167, 378-388. [9] Blake, C.C.F., Mair, G.A., North, A.C.T., Phillips, D.C. and Sarma, V.R.(1967) Proc. R. Soc. London B 167, 365-377. [10] Morosoli, R., Roy, C. and Yaguchi, M. (1986) Biochim. Biophys. Acta. 870, 473-478. [11] Bray, M.R. and Clarke, A.J. (1990) Biochem. J. 270, 91-96. [12] Ko, E.P., Akatsuka, H., Moriyama, H., Shinmyo, A., Hata, Y., Katsube, Y., Urabe, I. and Okada, H. (1992) Biochem. J. 288, 117-121. [13] Deshpande, V., Hinge, J. and Rao, M. (1990) Biochim. Biophys. Acta 1041, 172-177. [14] Keskar, S.S., Srinivasan, M.C. and Deshpande, V.V. (1989) Biochem. J. 261, 49-55. [15] Dey, D., Hinge, J., Shendye, A. and Rao, M. (1992) Can. J. Microbiol. 38, 436-441. [16] Hinge, J., Shendye, A., Srinivasan, M.C. and Rao, M. (1989) Patent application no.796-DEL-89 (7 Sept. 1989). [17] Chauthaiwale, J. and Rao, M. (1993) Biochem. Biophys. Res. Commun. 191, 922-927. [18] Miller, G.L. (1959) Anal. Chem. 31, 426-428 [19] Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. [20] Spackman, D.H., Stein, W.H. and Moore, S. (1958) Anal. Chem. 30, 1190-1206. [21] Hirs, C.H.W. (1967) Methods Enzymol. 11, 59-62. [22] Goodwin, T.W. and Morton, R.A. (1946) Biochem. J. 40, 628632. [23] Cavallini, D., Graziani, M.T. and Dupre, S. (1966) Nature (London) 212, 294-295. [24] Levy, H.M., Leber, P.D. and Ryan, E.M. (1963) J. Biol. Chem. 238, 3654-3659. [25] Sinha, U. and Brewer, J.M. (1985) Anal. Biochem. 151,327-333. [26] Clark, A.J. (1990) Biochim. Biophys. Acta 1040, 145-152. [27] Koyama, J., Inokuchi, N., Iwama, M. and Irie, M. (1985) J. Biochem. 97, 633-641. [28] Inoue, M., Yamada, H., Yasukochi, T., Kuroki, R., Miki, T., Horiuchi, T. and Imoto, T. (1992) Biochemistry 31, 5545-5553. [29] Parson, S.M. and Raftery, M.A. (1972) Biochemistry 11, 16231629. [30] Bastawde, K.B., Tabatabai, L.B., Meagher, M.M., Srinivasan, M.C., Vartak, H.G., Rele, M.V. and Reilly, P.J. (1991) ACS Symp. Ser. 460, 417-425.
ELSEVIER
Biochimica et Biophysica Acta 1204 (1994) 164-168
Biochi~ic~a et Biophysicat~ta
Chemical modification of xylanase from alkalothermophilic Bacillus species: evidence for essential carboxyl group 1 Jyoti Chauthaiwale, Mala Rao
*
Division of Biochemical Sciences, National Chemical Laboratory, Pune - 411008, India
(Received 29 April 1993; revised manuscript received 7 September 1993)
Abstract
The role of carboxyl group in the catalytic action of xylanase (M r 35 000) from an alkalothermophilic Bacillus sp. was delineated through kinetic and chemical modification studies using Woodward's Reagent K. The kinetics of inactivation indicated that one carboxyl residue was essential for the xylanase activity with a second order rate constant of 3300 M - 1 min- l. The spectrophotometric analysis at 340 nm revealed that the inhibition was correlated with modification of 24 carboxyl residues. In the presence of protecting ligand, modification of one carboxyl group was prevented. The pH profile showed apparent pK values of 5.2 and 6.4 for the free enzyme and 4.9 and 6.9 for enzyme-substrate complex. The pH dependence of inactivation was consistent with the modification of carboxyl group. The kinetic analysis of the modified enzyme showed similar K m and lower kca t values than the native enzyme indicating that catalytic hydrolysis and not the substrate binding was affected by chemical modification. The chemical modification of xylanase from alkalothermophilic Bacillus revealed the presence of tryptophans in the active site (Deshpande, V, Hinge, J. and Rao, M. (1990) Biochim. Biophys. Acta 1041, 172-177). This finding and present studies demonstrated the experimental evidence for the participation of carboxyl as well as tryptophan groups as essential residues of xylanase from alkalothermophilic Bacillus sp. Key words: Chemical modification; Xylanase; Alkalothermophilicity; Essential carboxyl group; (Bacillus sp.)
1. Introduction
Xylanase (1,4-/3-D-xylanohydrolase, EC 3.2.1.8) catalyzes the random hydrolysis of xylan to xylooligosaccharides and xylose which are useful feed stock for generating food and fuel [1]. Xylanases in conjunction with cellulases are useful for the complete conversion of cellulosic biomass to sugars [2]. The use of cellulase-free xylanases for the selective hydrolysis of the hemicellulose components in p a p e r and pulp has also received considerable attention [3]. Investigations on the roles of critical amino-acid residues of an enzyme are important in structure function analysis and protein engineering studies. The chemical modification and kinetic analysis of carbohydrases such as amylases [4,5] and cellulases [6,7] have revealed that tryptophan and carboxyl groups are involved in the catalytic activ-
* Corresponding author. Fax:+ 91212 334761. 1 NCL Communication No. 5766. 0167-4838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4838(94)E0189-9
ity. The involvement of carboxyl groups for the catalysis [8] and the participation of tryptophan residues for the substrate binding [9] have been shown for lysozyme. The studies on sequence similarities of xylanase, cellulase and lysozyme have supported the possibility that catalytic reaction of these enzymes follows the same mechanistic pathway [10]. Inspite of tremendous biotechnological applications of xylanases, little information is available on the mechanism of catalysis. However, there is one report on chemical modification of xylanase from fungus SchizophyUum c o m m u n e indicating the involvement of carboxyl groups in the catalysis [11]. Site-directed mutagenesis of xylanase from Bacillus p u m i l u s has shown that Glu-93 and Glu-182 are the essential catalytic residues [12]. The participation of tryptophan and cysteine residues at the active site of xylanases from Chainia [13] and Streptomyces T 7 [14] has been reported. The chemical modification studies showed the involvement of tryptophan residues at the catalytic site [13] of the xylanase (M r 35000) from alkalothermophilic Bacillus sp. [15,16]. The analysis of
J. Chauthaiwale,M. Rao / Biochimica et BiophysicaActa 1204 (1994) 164-168
the competitive inhibition of the xylanase (M r 35 000) by guanidine hydrochloride has shown the specific interaction between 'arginine like' guanidine moiety and carboxyl group at the active site of the enzyme [17]. The present paper describes the kinetic analysis and chemical modification studies that provide experimental evidence for the participation of essential carboxyl group in the catalytic mechanism of the xylanase of alkalothermophilic Bacillus. Our earlier findings [13] and present studies demonstrated the evidence for the presence of carboxyl as well as tryptophan groups as the essential residues of xylanase from alkalothermophilic Bacillus.
2. Materials and methods
2.1. Materials
Oat spelt xylan, standard amino-acid mixture, Woodward's Reagent K (2-ethyl-5-phenyl isoxazolium3-sulfonate) [WRK], dithionitrobenzoic acid (DTNB) and ethylamine were purchased from Sigma (USA). All other chemicals were of analytical grade. 2.2. Production and purification of enzyme
Alkalothermophilic Bacillus was grown in 250 ml flasks with 50 ml medium containing wheat bran (10%), yeast extract (1%) and Na2CO 3 (1%). The culture was incubated at 50°C and pH 10.0 for 48 h. The enzyme was purified by alcohol precipitation, gel filtration and preparative polyacrylamide gel electrophoresis as described earlier [15]. 2.3. Enzyme assay
The suitably diluted enzyme in 50 mM potassium phosphate buffer (pH 7.0) was incubated with substrate 1% xylan in a final volume of 1.0 ml at 50°C for 30 min. The reducing sugar released was determined by Miller's method [18] using D-xylose as standard. The unit of xylanase was defined as the 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 Bradford et al [19]. 2.4. Amino-acid composition
The amino-acid composition of the xylanase was performed in Spinco model 120-B automatic amino-acid analyzer by the method of Spackman et al. [20]. Protein (1 mg) was hydrolyzed for 24 h, 48 h and 72 h at ll0°C with 6 M HC1 in tubes that had been sealed under vacuum. Total cysteine content was determined by
165
performic acid oxidation [21]. The tryptophan and tyrosine contents were determined by the method of Goodwin and Morton [22]. Reduction of disulfide linkages with NaBH 4 in 8 M urea and subsequent titration with DTNB were performed [23]. 2.5. Modification by WRK
Xylanase (15 /xg) in 50 mM potassium phosphate buffer (pH 6.0) was incubated with different concentrations of WRK (5-30 mM) at 25°C. Aliquots of reaction mixture were withdrawn at indicated time intervals and excess reagent was quenched with sodium acetate buffer (pH 5.0) (final concentration 250 mM). The residual xylanase activity was measured and expressed as percentage of a control. Control tubes having enzyme or inhibitor with and without substrate were incubated under identical conditions. The pseudo-firstorder rate constants were obtained from the slopes of the plots of logarithm of the residual activity against time. The second order rate constant was calculated from the slope of the plot of pseudo-first-order rate constants against concentration of inhibitor [24]. Xylanase was incubated with different concentrations of WRK (1-60 mM) for 30 min, the reaction was quenched with sodium acetate buffer (pH 5.0) and the samples were passed through Bio-Gel P-10 column equilibriated and eluted (1 m l / 2 min) with 50 mM potassium phosphate buffer (pH 6.0). Aliquots were removed to estimate the residual activity. The degree of modification was measured by the increase in absorbance at 340 nm (7000 M -1 cm -1) [25]. The modification of xylanase in presence and absence of xylose (20 mM) was performed similarly with the estimation of residual activity. The enzyme modified with WRK (50 mM, 30 min) was hydrolyzed for 24 h and analyzed for amino-acid residues as described earlier in this section. Ethylamine and standard amino-acid mixture were used as standards.
3. Results and discussion
3.1. Amino-acid analysis
Amino-acid analysis of the xylanase from alkalothermophilic Bacillus showed predominance of acidic amino acids and glycine (Table 1). Spectrophotometric analysis of protein sample by Goodwin and Morton method indicated the presence of 10 tryptophan residues per mole of enzyme and the ratio of tryptophan to tyrosine was found to be 5:1. Analysis of sample oxidized with performic acid showed the presence of one cysteine residue per enzyme molecule. Presence of one SH group was also indicated by the
J. Chauthaiwale, M. Rao / Biochimica et Biophysica Acta 1204 (1994) 164-168
166
Table 1 Amino-acid composition of xylanase from alkalothermophilic Bacillus Amino acid
Gly Ala Thr Ser Pro Asx Glx His Lys Arg Val Ile Leu Met Trp Tyr Phe Cys
Composition (residues/mol of enzyme)
mole%
98 4 2 2 1 66 158 1 2 1 1 2 2 3 10 2 1 1
27.88 + 0.10 1.00 + 0.08 0.44 + 0.10 0.46 + 0.06 0.17 + 0.03 18.98 + 0.10 45.00 + 0.12 0.18 + 0.02 0.51 + 0.04 0.21 + 0.03 0.23 + 0.07 0.43 + 0.06 0.44 + 0.04 0.76 + 0.10 2.72 + 0.20 0.43 + 0.01 0.15 + 0.01 0.23 + 0.01
DTNB titration of denatured xylanase. Analysis of reduced enzyme indicated the absence of disulfide bridge.
3.2. Chemical modification of xylanase by WRK The reaction of the enzyme with the carboxyl group specific Woodward's Reagent K resulted in progressive loss of activity. When enzyme was incubated with 20 mM WRK for 30 rain the extent of inactivation observed was 90%. Inactivation of xylanase by WRK was dependent upon the time and reagent concentration but the pattern of inhibition was complex as shown by the biphasic nature of semilogarithmic plots of residual activity as a function of time (Fig. 1). The course of inhibition can be resolved into two first order processes with the slope at longer times determining the rate of inactivation. The biphasic nature of the kinetics of inactivation can be explained on the basis of apparently large number of carboxyl residues exposed to the action of the WRK. The initial inactivation of the enzyme might be due to the modification of readily available non-catalytic carboxyl groups which might cause slight alteration in the microenvironment of the active site. During the second phase the modifier specifically reacts with the carboxyl groups located at the active site causing time- and concentration-dependent inhibition. The biphasic nature of inactivation is a common phenomenon among the carboxyl group modification of a variety of enzymes including carbohydrases [26,27]. A double-logarithmic plot of the observed pseudo-first-order rate constants against reagent concentration yielded an order of 1.0 indicating that
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Fig. 1. Kinetics of inactivation of xylanase from alkalothermophilic Bacillus: Xylanase (15/xg) was incubated with WRK 5 mM (o), 10 mM ( x ) , 20 mM (zx) and 30 mM ([]) W R K at 25°C. Aliquots of reaction mixture were removed at indicated time-intervals. The residual xylanase activity was measured and expressed as percentage of control. Inset: Apparent order of reaction with respect to reagent concentration: The pseudo-first-order rate constants (k) were calculated from the slopes of semilogarithmic plots of residual activity against time.
modification of a single carboxyl residue resulted in the loss of enzyme activity (Fig. 1 inset). The second order rate constant was calculated to be 3300 M-1 min-1. The reaction of WRK is initiated by the formation of ketoketenimine which modifies the carboxyl group of the enzyme to give an enol ester with concomitant increase in the absorbance at 340 nm [25]. The spectral analysis of WRK modified xylanase at 340 nm revealed the modification of 24 carboxyl residues per molecule of the enzyme. However in the presence of xylose, the competitive inhibitor of the xylanase, 23 carboxyl residues per molecule of the enzyme were modified indicating the presence of a single carboxyl group at the active site (Table 2). The possibility of interference of sulfhydryl groups in the spectral analysis can be excluded, since DTNB titration of native xylanase did not show any change in the absorbance indicating the absorbance at 340 nm was entirely owing to carboxyl group modification. Fig. 2 shows the residual enzymatic activity plotted against the number of carboxyl groups modified. The Table 2 Differential modification of xylanase by WRK in presence and absence of xylose, a competitive inhibitor No.
Sample
Residual activity (%)
Reading at 340 nm
No. of carboxyl groups modified
1 2 3
Enzyme Enzyme + WRK Enzyme + xyiose + W R K
100 20 80
0.018 0.523 0.506
24.04 23.20
Xylanase (100 /zg) was modified with WRk (50 mM, 30 min) in presence or absence of 20 mM xylose.
J. Chauthaiwalei M. Rao / Biochirnica et Biophysica Acta 1204 (1994) 164-168
167
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Fig. 3. Determination of K m and kcat for native xylanase ( × ) and xylanase mofidied by WRK (<3). The native and partially modified (10 mM WRK for 10 min) enzymes were allowed to react with different concentrations of xylan (1-10 mg) and the activity was estimated. The values for K m and Kcat were determined from Lineweaver-Burk plot.
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Fig. 2. Plot of percentage residual activity against number of carboxyl groups modified. Xylanase (100 tzg) was incubated with different concentrations of WRK (1-60 mM) for 30 min. The residual activity was determined and the samples were analyzed spectrophotometrically at 340 nm. The number of carboxyl groups modified was determined by assuming molar absorption coefficient of 7000 M-1 cm-
1.
extrapolation with the Y- intercept passing through 0% inactivation showed the presence of one carboxyl group at the catalytic site. A comparison of the amino-acid composition of the unmodified and WRK modified xylanase revealed that contents of amino-acid residues other than Asx and Glx were not altered (data not shown).The modification of carboxyl residues was confirmed by the liberation of ethylamine [25].
5.2 and 6.4 for the free enzyme and 4.9 and 6.9 for the enzyme-substrate complex suggesting the protonated forms of aspartic a n d / o r glutamic acid. The pH oriented inactivation and the absence of histidyl group at the active site [13] indicated the ionizable groups as carboxylates. The pK value of the xylanase towards neutrality can be ascribed to a carboxyl group. The higher pK values of glutamic acid, as observed in case of lysozyme, is attributed to the fact that the residue is located in a non-polar environment [28]. The presence of tryptophan at the active site may create a hydrophobic environment. Ionization of a neighboring acidic residue has been shown to increase the pK value. The binding of substrate, especially long-chain substrate, induces local conformational changes that alters the microenviron-
3.3. Kinetic analysis: pH dependence The kinetic parameters Michaelis-Menten constant (K m) and turnover number ( k c a t) w e r e obtained from Lineweaver-Burk plots of the data (Fig. 3). The K m of WRK modified enzyme was found to be comparable (1.81 mg) to that of native enzyme (1.58 mg) at pH 6.0. However, the catalytic rate constant of modified enzyme was calculated to be 2.13.10 -3 S -1 which was three orders of magnitude lower than the native xylanase (6.17.10 -3 S -1) indicating that the catalytic hydrolysis and not the substrate binding is affected by the chemical modification. The inactivation of xylanase by WRK was found to be pH dependent and maximum inhibition occurred at pH 6.0 (Fig. 4). The plot of log K against pH showed a bell-shaped graph indicating the presence of two ionizable groups with pK values 5.3' and 6.4. Semilogarithmic plots of kcat and kcat.Km 1 against pH are shown in Fig. 5. These profiles indicated apparent pK values of
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pH Fig. 4. pH-dependent WRK modification of xylanase: Xylanase (15 /.~g) was modified by WRK (10 mM) in 50 mM buffers of different pH (pH 4 and 5, KCI/HCI; pH 6-8 potassium phosphate). Aliquots were removed after indicated time-intervals and were checked for xylanase acitivity. The pseudo-first-order rate constants were obtained from the slopes of the plots of logarithm of residual activity against time.
168
J. Chauthaiwale, M. Rao / Biochimica et Biophysica Acta 1204 (1994) 164-168 ,¢
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References
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ship a w a r d e d by t h e University G r a n t s C o m m i s s i o n to Jyoti V. C h a u t h a i w a l e a r e gratefully a c k n o w l e d g e d .
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Fig. 5. Dependence of kinetic parameters on pH: Xylanase (3 /zg) was reacted with different concentrations of substrate (1-10 mg of soluble xylan) in 50 mM buffers of pH ranging from 4-8. K m and Kca t values were derived from Lineweaver Burk plots. Effect of pH on (a) log kca t (b) log kcat.Km1.
m e n t o f acidic a m i n o - a c i d r e s i d u e resulting in an inc r e a s e in its p K v a l u e [29]. I n case o f lysozyme t h e high p K value o f Glu-35 is c o n s i d e r e d to b e critical for the c a r b o x y l a t e g r o u p to act at h i g h e r p H [28]. T h e xylanase f r o m a l k a l o t h e r m o p h i l i c Bacillus s h o w e d optim u m activity at p H 6.0 b u t was also active at h i g h e r p H [15] which m a y b e d u e to t h e h i g h e r p K value o f e s s e n t i a l carboxyl group. T h e p r e s e n c e o f carboxyl r e s i d u e at t h e active site of xylanase as i n d i c a t e d in t h e p r e s e n t studies is consist e n t with t h e o b s e r v a t i o n t h a t a s p a r t i c acid r e s i d u e involved in t h e catalysis is c o n s e r v e d a m o n g m i c r o b i a l g l y c o h y d r o l a s e s [30].
Acknowledgments
T h e p a r t i a l s u p p o r t by t h e D e p a r t m e n t o f Biotechnology ( G o v e r n m e n t o f I n d i a ) a n d t h e r e s e a r c h fellow-
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