Isabel DE LA MATA, Pilar ESTRADA, Ricardo MACARRON, Juan Manuel DOMINGUEZ,. Ma Pilar CASTILLON and Carmen ACEBAL*. Departamento de ...
679
Biochem. J. (1992) 283, 679-682 (Printed in Great Britain)
Chemical mechanism of fl-glucosidase from Trichoderma reesei QM 9414 pH-dependence of kinetic parameters Isabel DE LA MATA, Pilar ESTRADA, Ricardo MACARRON, Juan Manuel DOMINGUEZ, Ma Pilar CASTILLON and Carmen ACEBAL* Departamento de Bioquimica
y
Biologia Molecular
I,
Facultad de Quimica, Universidad Complutense, 28040 Madrid, Spain
The variation of kinetic parameters of /I-glucosidase from Trichoderma reesei QM 9414 with pH was used to gain information about the chemical mechanism of the reaction catalysed by this enzyme. The pH-dependence of Vmax and Vmax /Km for p-nitrophenyl /J-D-glucopyranoside showed that a group with a pK value of 4.3 must be unprotonated and a group with a pK value of 5.9 must be protonated for activity. Temperature and solvent-perturbation studies indicated that these groups are a histidine residue and a carboxy group respectively. Profiles of pK1 for maltose as competitive inhibitor showed that binding is prevented when a group on the enzyme with a pK value of 4.5 becomes protonated.
INTRODUCTION
MATERIALS AND METHODS
It is well known that ,-glucosidase (EC 3.2.1.21) plays an important role in the enzymic hydrolysis of cellulose by stopping cellobiose from accumulating and inhibiting other cellulases. Over the past few years, many workers have studied the mechanism of cellulose breakdown in order to obtain fermentable sugars at low cost. However, optimization of the process has become a challenge because of the difficulty of hydrolysing a polymer with structural characteristics that represent an important barrier for both chemical and enzymic hydrolysis. The enzyme ,-glucosidase is one component of the cellulase system complex. It catalyses cleavage of cellobiose and other cello-oligosaccharides to yield D-glucose as the final product of enzymic hydrolysis of cellulose. Whereas its activity has been exhaustively investigated and purification methods have been recently described, relatively little use has been made of the steady-state kinetics in determining the groups on the enzyme responsible for substrate binding and catalysis. Available evidence suggests that the essential catalytic groups in lysozymes, ,6-glucosidases and other glucosidases are two carboxy groups (Sinnott, 1987); however, the presence of other groups with catalytic competence cannot be ruled out (Umezurike, 1977; Bronnenmeier & Staudenbauer, 1988). The catalytic mechanism of enzymes that hydrolyse glucosides has been studied for several years (Koshland, 1953; Sinnott, 1987). Some researchers point to the involvement of a stabilized oxo-carbonium ion intermediate in a mechanism like that proposed for lysozyme (Withers & Street, 1988). However, since /I-glucosidase hydrolyses glucosides with retention of anomeric configuration by a double-displacement mechanism (Withers & Street, 1988; Estrada et al., 1990), the existence of a covalent glycosyl-enzyme cannot be excluded. The present paper reports evidence about the chemical mechanism of /J-glucosidase from Trichoderma reesei QM 9414 by examining the pH-dependence of kinetic constants of the enzyme and the variation with pH of K1 for maltose as competitive inhibitor.
Chemicals Maltose and p-nitrophenyl-,/-D-glucopyranoside (pNPG) were from Fluka (Buchs, Switzerland); Mes was from Sigma Chemical Co. (St. Louis, MO, U.S.A.). All other reagents and solvents were analytical-reagent grade from Merck (Darmstadt, Germany).
Abbreviation used: pNPG, p-nitrophenyl f-D-glucopyranoside. * To whom correspondence should be addressed.
Vol. 283
Enzyme purification ,8-Glucosidase from Trichoderma reesei QM 9414 was purified as previously described (Estrada et al., 1990). Kinetic assays The activity of ,-glucosidase was determined in incubation mixtures containing 25 ,ug of enzyme and 5 mM-pNPG in 2 ml of 0.1 M-sodium citrate buffer, pH 4.8, at 50 'C for 10 min. The release of p-nitrophenol was measured at 420 nm, in a Beckman DU-70 computerized spectrophotometer, after the addition of 3 ml of 0.1 M-NaOH. Activity was linear up to 90 jug of protein and up to 90 min under the stated conditions. The pH-dependence of the kinetic parameters was determined in buffers of different pH (4.0-6.5, sodium citrate/sodium phosphate mixture), in the temperature range 30-60 'C. Solvent-perturbation studies with 20 (v/v) propan-2-ol were performed in neutral acid buffer (0.1 M-sodium citrate/sodium phosphate) as well as in cationic buffer (0.1 M-Mes). The organic solvent was added after determination of the pH in the assay mixture. Propan-2-ol (20 %) caused no loss of enzyme activity. All pH measurements were performed with a Radiometer 26 pHmeter standardized at the given temperature to + 0.03 pH unit with Beckman buffers. The pH-dependence of K, was examined for maltose as competitive inhibitor of ,-glucosidase (Estrada et al., 1990). Assays were run at variable concentration of pNPG (from 0.1 to 5 mM) with maltose varied from 0 to 100 mm, at 50 'C for 10 min. All the experiments were carried out in triplicate. In Figures and Tables, the mean value of the triplicates is represented.
I. de la Mata and others
680
(a)
(a)
0.2
6.0 p
0
0
5.5 I
0.0 )i
E
5.0
0
-0.2 F
4.5
0
F
4.0
-0.4 -
4.0
4.5
6.0
5.5
5.0
3.0
6.5
3.3
103/T(K-1)
pH 1.0
3.2
3.1
6
(b)
-
(b)
0
5
:E 0.8
0
0~
E
° 0.6
< Es-I
F
4
--1~
3.0
0.4
5.0
4.5
4.0
5.5
6.0
6.5
pH
Fig. 1. pH variation of log V..x. (a) and log(V ,,.a/Km) (b) Sodium citrate/sodium phosphate (0.1 M) was used as wide-pHrange buffer. The incubation mixtures contained 50 ,ul of enzyme solution (0.5 mg/ml), 1 ml of substrate solution (substrate concentration was varied from 0.1 mm to 5 mM) and 0.95 ml of buffer. Incubations were carried out for 10 min at 50 'C. The enzyme was stable in the pH range assayed.
Statistical analysis of the data Values of kinetic constants were determined by fitting initialvelocity data to the Woolf-Augustinsson-Hofstee equation (Segel, 1975) by a weighted least-squares method developed in our laboratory. Substrate saturation curves were fitted to eqn. (1). Competitive inhibition data with maltose were fitted to eqn. (2).
,,
v
=
,
Vmax,S/(Km+ S) ,
Vmax. S/[Km(l + I/Kj) + S obtained by fitting pH profiles to
(1I)
v=
pK values were using the BMDP program: y
=
C/[l + (H/K1) + (K2/H)]
eqn.
(3) by (3) de-
The enthalpies of ionization of enzyme residues were termined by fitting the observed pK values from pH profiles obtained at different temperatures to eqn. (4): (4) pK = AH10n/(2.3RT) calculated was inhibitor by as maltose competitive for pK, fitting the data from the corresponding pH profiles to eqn. (5): y
=
C/f[
+ (H/K1)]
(5)
In eqns. (1) and (2), v and Vmax are initial and maximum velocity respectively, Km is the Michaelis constant, S is the
3.2
3.1
3.3
103/T(K-1) Fig. 2. Temperature-dependence of pK values from V... (a) and V.ax IK. (b) profiles Experiments similar to those of Fig. 1 were carried out at four temperatures and the corresponding pK values were calculated. Symbols: *, pKA; 0, pK2.
substrate concentration, I is the inhibitor concentration and K1 is the inhibition constant. In eqns. (3) and (5), y is the corresponding constant, C is the value of y attained at the optimum state of protonation, H is the hydrogen-ion concentration, and K1 and K2 are acid dissociation constants of groups that must be deprotonated and protonated respectively for activity. In eqn. (4) AHion is the enthalpy change on dissociation, R the gas constant and T the absolute temperature.
RESULTS The pH-dependence of the maximum velocity (V..x.) and specificity constant (Vmax /Km) with pNPG as substrate is shown in Fig. 1. Both kinetic constants decreased below pH 4.5, with a limiting slope close to + 1, indicating that protonation of a single group causes loss of activity. V.ax. and VmJax/Km also decreased above pH 5.2 with a limiting slope close to -1, indicating that deprotonation of a single group causes loss of activity. The data 4.3 and pK2 5.9. were fitted to eqn. (3) giving pK1 To determine the identity of the groups responsible for these pK values, the temperature-dependence of the VKax and Vmax /Km profiles was examined. Experiments were carried at 30, 40, 50 and 60 °C (Fig. 2). No irreversible thermal inactivation was observed at these temperatures. The data were fitted to eqn. (4) to determine the corresponding ionization enthalpies (AHio.): AHion = 27.2 kJ/mol for pK, and AHion =-6.5 kJ/mol for pK2 from V profiles =
=
1992
Chemical mechanism of ,-glucosidase
681
Table 1. Effect of 20 % (v/v) propan-2-ol on pK values calculated from proffles of log( VmaxIKm) and log V.a. versus pH, in either neutral or cationic acid buffers pK values and their standard deviations were obtained by statistical analysis of the parameter data in accordance with eqn. (3).
Neutral buffer
Cationic buffer
-Propan-2-ol
+ Propan-2-ol
-Propan-2-ol
+ Propan-2-ol
4.16+0.24 5.40+0.20
3.33 +0.25 5.41 +0.08
3.41 +0.25 6.23 +0.06
3.40+0.11 6.24 + 0.05
4.18 +0.21 5.89 +0.18
3.40+0.17 5.97 +0.07
3.17+0.28 6.49+0.13
3.33 +0.14 7.09+0.13
log Vmax. pK1
pK2 log( Vmax./Km) pKA pK2
cationic acid type. Since the substrate has no ionizable group with this pK value, it follows that deprotonation of a residue on the enzyme is responsible for binding and catalysis.
-1.4 0~~~
-1.6
-1.8
-2.0
I 4.0
4.5
5.0
5.5
6.0
6.5
pH Fig. 3. Variation of pK; with pH for maltose Assays
were
carried out
as
in
Fig.
with maltose varied from 0 to
100mmM.
AHion = 26.9 kJ/mol for pK1 and AHi.n= 16.5 kJ/mol for -
pK2 from V/K profiles
These values of AH10n point to the ionization of a histidine residue and a carboxy group as being responsible for the decrease in activity at low and high pH respectively (Cleland, 1977). Further evidence about the nature of the groups responsible for these pK values was obtained by examining the effect of the addition of organic solvents on the pK in neutral and cationic buffers (Table 1). Addition of 200% (v/v) propan-2-ol caused a decrease in the pK, value observed in neutral acid buffer, whereas this pK remained unchanged in cationic acid buffer, confirming the identity of the group as being of the cationic acid type. On the other hand, the addition of 20 %O propan-2-ol caused no change in the pK2 value observed in neutral acid buffer, whereas it induced an increase in this pK in cationic acid buffer, indicating that the catalytic group is of the neutral acid type. Profiles of pK, detect groups with protonation states that affect binding. A group necessary for catalysis but not directly participating in the binding process will not show up, or will show only a small change in affinity with change in protonation state (Cleland, 1982). The pH-dependence of Ki was examined for maltose as competitive inhibitor of pNPG. Analysis of the data of the pKi profile (Fig. 3, eqn. 5) yielded a pK, value of 4.5, indicating that a group with a pK of 4.5 must be unprotonated for the substance to bind. The marked change in slope in the profile corresponds to that seen in the V1max/.Km and Vmax profiles, which were described above as demonstrating a group of the Vol. 283
DISCUSSION In recent years a great controversy has emerged about the catalytic mechanism of enzymes that hydrolyse ,J-glucosidic bonds with retention of anomeric conformation (Sinnott, 1987; Withers, 1988; Clarke, 1990). Hydrolytic reactions catalysed by glycosidases resemble the acid-catalysed hydrolysis of alkyl glycosides or aryl glycosides in which the cleavage of the glycosyl (C-1-O) bond occurs. Therefore these reactions are nucleophilic substitutions at C-I of the glycoside (Koshland, 1953; Donsimoni et al., 1988). The first step should be protonation of the anomeric oxygen atom by an acidic group of the enzyme to give the aglycone moiety of the substrate. The glycosyl-enzyme intermediate could be either a stabilized carbocation or a covalent intermediate. Recent studies point to a covalent glycopyranosyl intermediate (Withers & Street, 1988). In a second step, a group of the enzyme is involved in a general base catalysis, and a hydroxy group is stereospecifically added to the glycosyl moiety of the substrate. Water, alcohols or some other hydroxy compound can be involved as hydroxy-group donor. With respect to the amino acid residues essential for the enzyme reaction catalysed by ,-glucosidase, several authors have pointed to the presence of two essential carboxy groups at the active site of the enzyme, suggesting a mechanism that resembles that described for lysozyme (Blake et al., 1968; Donsimoni et al., 1988; Clarke, 1990). Kinetic and chemical-modification studies of the enzyme from Botryodiplodia theobromae have shown the participation of a carboxy group and a histidine residue at the catalytic centre (Umezurike, 1977, 1987). Both types of amino acid residue have also been suggested to play a catalytic role in fl-glucosidase from almonds (Hsuanyu & Laidler, 1984). The plot of log (JVm'. /Km) for pNPG as a function of pH shows marked changes in slope at both high and low pH (Fig. 1). The one at low pH corresponds to a pK value of 4.3 and has a limiting slope close to + 1. Therefore a group in the substrate or in the enzyme must be unprotonated for activity. As pNPG has no ionizable group with this pK value, it follows that a group on the enzyme is essential for activity. The results of the solventperturbation experiments (Table 1) and a AHion value of 27.2 kJ/mol point to this group being a histidine residue (Cleland, 1977). The change in slope in the VJ.ax./Km profile at higher pH (Fig. 1) corresponds to a pK value of 5.9 and has a limiting slope close to -1. As the substrate contains no ionizable group with this pK
682 value, a group on the enzyme that must be protonated is essential for activity. The results of the solvent-perturbation experiments (Table 1) and the AH10. value of -6.5 kJ/mol indicate that this group is a neutral acid. The data are thus consistent with the presence of both a histidine residue and a carboxy group at the active site of /J-glucosidase, which are responsible for binding, for catalysis or for both. The binding of pNPG is prevented when a group with a pK value of 4.5 becomes protonated (Fig. 3). This pK value is similar to the lower pK value of 4.3 from the Vm.ax /Km profile, suggesting the involvement of the histidine residue in the binding of the substrate. The absence of a break in the pKA profile at higher pH points to the carboxy residue as being responsible only for catalysis. If the histidine were also involved with catalysis, its ionization should be observed in the Vmax. profile. The Vm.. profile (Fig. 1) shows that maximum enzyme activity is achieved when a group with a pK of 4.3 becomes unprotonated and when a group with a pK of 5.9 becomes protonated. Therefore the ionization of the same group is being observed in the Vm1ax/K, pKi and Vm.ax profiles at the lower pH, indicating that the histidine residue is responsible for binding and catalysis. Since the pK observed for histidine has the same value in the Vm1.' and Vmax /Km profiles, it follows that the binding of the substrate is not of an ionic type (between positively and negatively charged groups), but, since the substrate prefers to bind to one protonation state of the enzyme, the involvement of the histidine residue in hydrogenbonding with the substrate cannot be discounted (Cleland, 1982). The evidence presented in this paper may be interpreted in terms of a chemical mechanism for catalysis. Since recent results
I. de la Mata and others
point to a covalent glycopyranosyl intermediate in the reaction catalysed by ,3-glucosidase (Withers & Street, 1988), the existence could be inferred of an interaction between the histidine residue and a nucleophilic residue in the active centre (probably a cysteine or a serine residue), enhancing the nucleophilicity of this hypothetical essential residue. The catalytic carboxy group could be involved in the aglycone departure of the substrate, acting as a general base to increase the nucleophilicity of a water molecule. REFERENCES Blake, C. C. F., Johnson, L. N., Mair, G. A., North, A. T. C., Phillips, D. C. & Sarma, V. R. (1968) Proc. R. Soc. London B 167, 378-385 Bronnenmeier, K. & Staudenbauer, W. L. (1988) Appl. Microbiol. Biotechnol. 28, 380-386 Clarke, A. J. (1990) Biochim. Biophys. Acta 1040, 145-152 Cleland, W. W. (1977) Adv. Enzymol. Relat. Areas Mol. Biol. 45, 273-387 Cleland, W. W. (.1982) Methods Enzymol. 87, 390-405 Donsimoni, R., Legler, G., Bourbouze, R. & Lalegerie, P. (1988) Enzyme 39, 78-89 Estrada, P., Mata, I., Dominguez, J. M., Castill6n, M. P. & Acebal, C. (1990) Biochim. Biophys. Acta 1033, 298-304 Hsuanyu, Y. & Laidler, K. J. (1984) Can. J. Biochem. Cell. Biol. 63, 167-175 Koshland, D. E. (1953) Biol. Rev. Cambridge Philos. Soc. 28, 416-436 Segel, I. M. (1975) Enzyme Kinetics, pp. 210-214, Wiley-Interscience, New York Sinnott, M. L. (1987) in Enzyme Mechanism (Page, M. I. & Williams, A., eds.), pp. 259-297, The Royal Society of Chemistry, London Umezurike, G. M. (1977) Biochem. J. 167, 831-833 Umezurike, G. M. (1987) Biochem. J. 241, 455-462 Withers, S. G. & Street, I. P. (1988) J. Am. Chem. Soc. 110, 8551-8553
Received 30 July 1991/8 October 1991; accepted 21 October 1991
1992
