Biochem. J. (1989) 261, 819-825 (Printed in Great Britain)

Fungal cellulase systemsComparison of the specificities of the cellobiohydrolases isolated from Penicillium pinophilum and

Trichoderma reesei

Marc CLAEYSSENS,*t Herman VAN TILBEURGH,* Peter TOMME,* Thomas M. WOODtand Sheila I. McRAEt*Laboratorium voor Biochemie, Rijksuniversiteit Gent, B-9000 Gent, Belgium, and tRowett Research Institute, Bucksburn,Aberdeen AB2 9SB, U.K.

Reaction patterns for the hydrolysis of chromophoric glycosides from cello-oligosaccharides and lactose bythe cellobiohydrolases (CBH I and CBH II) purified from Trichoderma reesei and Penicillium pinophilumwere determined. They coincide with those found for the parent unsubstituted sugars. CBH I enzyme fromboth organisms attacks these substrates in a random manner. Turnover numbers are, however, low and donot increase appreciably as a function of the degree of polymerization of the substrates. The active-sitetopology of the CBH I from T. reesei was further probed by equilibrium binding experiments withcellobiose, cellotriose, lactose and some of their derivatives. These point to a single interaction site (ABC),spatially restricted as deduced from the apparent independency of the thermodynamic parameters. Itappears that the putative subsite A can accommodate a galactopyranosyl or glucopyranosyl group, andsubsite B a glucopyranosyl group, whereas in subsite C either a glucopyranosyl or a chromophoric groupcan be bound, scission occurring between subsites B and C. The apparent kinetic parameters (turnovernumbers) for the hydrolysis of cello-oligosaccharides (and their derivatives) by the CBH II type enzymeincrease as a function of chain length, indicative of an extended binding site (A-F). Its architecture allowsfor specific binding of ,-(1 -+4)-glucopyranosyl groups in subsites A, B and C. Binding of a chromophorein subsite C produces a non-hydrolysable complex. The thermodynamic interaction parameters of someligands common to both type of enzyme were compared: these substantiate the conclusions reached above.

INTRODUCTION

The presence of two immunologically unrelated cello-biohydrolases (CBH I and II, EC 3.2.1.91) in extracellularculture fluids of both Trichoderma sp. (Gritzali & Brown,1979; Fagerstam & Pettersson, 1979; Montenecourt,1983) and Penicillium pinophilum (Wood & McCrae,1980, 1982) has been well established. These enzymes aresimilar in their molecular masses (approx. 50 kDa), theirpl values (acidic range) and their capacities for actingsynergistically with reconstituted mixtures of endo-glucanases in solubilizing microcrystalline cellulose(Henrissat et al., 1985; Wood & McCrae, 1986). Fur-thermore, CBH I and CBH II from both species havebeen shown to act synergistically (Fagerstam &Pettersson, 1979; Wood & McCrae, 1986). The substratespecificities of CBH I and II have been studied mostly inrelation to their capacities to catalyse the hydrolysis ofsoluble and insoluble cellulose derivatives (Enari & Niku-Paavola, 1987). However, the chromophoric (fluoro-phoric) substrates described in the present study provedto be useful in earlier studies of CBH I, CBH II andendocellulases from Trichoderma reesei (van Tilbeurgh &Claeyssens, 1985), and to have some efficacy as probes ofthe active sites of the enzymes (van Tilbeurgh et al.,

1988). Thus these substrates have been used to provideinformation on structure-activity relationships of thecomponent enzymes of the cellulase complex and in thedifferentiation of two domains (i.e. catalytic and binding)in CBH I and CBH II from T. reesei (van Tilbeurghet al., 1986; Tomme et al., 1988). Successful crystal-lization of the 'core enzymes' containing the active-sitedomains from Trichoderma CBH I and CBH II augurswell for the release of detailed structural information ofthese enzymes in the not too distant future (A. Jones,personal communication).The present paper describes the parameters affecting

enzymic activity on some substrates in a comparativestudy of the cellobiohydrolases purified from T. reeseiand P. pinophilum. More detailed information on theactive-site topology of CBH I (T. reesei) was obtainedand is compared with the data collected in a previousstudy of CBH II (van Tilbeurgh et al., 1985).

EXPERIMENTAL

Purification of the enzymes

The CBH I and CBH II from Trichoderma reesei werepurified by affinity chromatography as reported by van

Tilbeurgh et al. (1984). By this procedure mixtures of

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Abbreviations used: CBH, cellobiohydrolase; EG, endoglucanase; MeUmb(Glc)n (n = 1-5), 4-methylumbelliferyl f-D-glycosides from glucose,cellobiose, cellotriose, cellotetraose and cellopentaose; MeUmb, 4-methylumbelliferone; MeUmbLac, 4-methylumbelliferyl /-D-glycoside fromlactose; N2PhSC, 2',4'-dinitrophenyl l-thio-,8-D-cellobioside; CNPhLac, 2'-chloro-4'-nitrophenyl /8-D-lactoside.

I To whom correspondence should be addressed.

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M. Claeyssens and others

iso-components were obtained that are enzymically in-distinguishable from the purified main fraction (pl 3.9for CBH I and pl 5.9 for CBH II). The molar absorptioncoefficients (280 nm) are 73 000 M-1. cm-' for CBH I and75000 M-'cm-' for CBH II (Tomme et al., 1988).The cellobiohydrolases from Penicillium pinophilum

were purified as described by Wood & McCrae (1980,1982) or alternatively by affinity chromatography (vanTilbeurgh et al., 1984). Properties of these enzymes are asreported previously (Wood, 1988).Substrates and ligands

Cellobiose and lactose were commercial products.Cello-oligosaccharides were prepared from cellulose(Whatman CC4 1) acetolysates and fractionation of thedeacetylated mixtures by gel filtration (Bio-Gel P-2) withwater as eluent (65 °C). Reducing-end-labelled (1-3H)sugars were fractionated from a cello-oligosaccharidemixture, prepared at the U.V.V.R. (Prague, Czechos-lovakia, courtesy of Dr. Peter Biely of Bratislava) bypreparative h.p.l.c. (see below). The specific radio-activities were 10.5 GBq/mmol and samples were dilutedwith non-labelled sugars as appropriate. 4-Methyl-umbelliferyl, 2'-chloro-4'-nitrophenyl and 2',4'-dinitro-phenyl 1-thioglycosides of cello-oligosaccharides andlactose were prepared as described previously (vanTilbeurgh et al., 1988).Enzyme assaysThe enzymic hydrolysis of the chromophoric substrates

was followed by h.p.l.c. (van Tilbeurgh et al., 1982). A25 cm x 0.46 cm RSil-Polyol 5,tm-particle-size column(Alltech)- and a Waters chromatographic system wereused [eluent acetonitrile/water (39: 11, v/v) at 1.5 ml/min). Samples (20,l) were injected (Valco) from areaction mixture (in 50 mM-sodium acetate/acetic acidbuffer, pH 5.0, at 25 °C) diluted 1:3 in acetonitrile. Theconcentration of chromophoric reaction products wasdetermined at 313 nm with a Waters 440 detector (peakheights). The unmodified cello-oligosaccharides were alsoanalysed by h.p.l.c. [same column, eluent acetonitrile/water 3:2 (v/v)] and monitored with a Waters 400refractive-index detector. Radioactive fractions (50-100 ,1) were collected and their radioactivities countedconventionally (Tri-Carb spectrometer) after additionof a scintillation fluid (4 ml of Aqualuma; Lumac).With MeUmb(Glc)2 and MeUmbLac as substrate

enzymic activities were monitored continuously byrecording the liberated MeUmb at 347 nm (Perkin-ElmerB55 spectrophotometer) or discontinuously by fluori-metry at pH 10 (Vitatron photometer, excitation at366 nm and emission at > 400 nm).

.

The 2'-chloro-4'-nitrophenyl derivatives were used in acontinuous assay (405 nm) at pH 5.7 (50 mM-sodiumacetate buffer), and the increase in concentration ofreaction product (2-chloro-4-nitrophenol) as a functionof time was calculated from the molar absorption coeffi-cient under these conditions (9500 M-1 cm-1).When cellotriose or one of the higher cello-

oligosaccharides was used as substrate, glucose wasdetermined by the glucose oxidase/peroxidase method(Bergmeyer, 1974).Binding experiments

Equilibrium dialysis was performed by forced-flowdiafiltration, as described previously (Claeyssens et al.,1985).

Difference absorption spectra were obtained with aUVICON 810 double-beam spectrophotometer (Kon-tron) and tandem mixing cuvettes (Yankeelov, 1963).Titrations were performed with two 1 cm x 1 cm x 4.5 cmcuvettes, equipped with motor-driven stirrers and con-nected to two Hamilton syringes that deliver a solutionof a non-binding sugar (sucrose) to the reference cuvetteand a solution of the appropriate ligand to the samplecuvette (De Boeck et al., 1984). The cuvettes were firstfilled with buffer and then the ligands were added. Theresulting difference spectrum was stored. The cuvetteswere then filled with enzyme solution and the netdifference spectra recorded after the addition of theligands. Difference absorption titrations were recordedat the maxima of the difference spectra. The results weretreated as described previously (De Boeck et al., 1984).

RESULTS

Specificity of cellobiohydrolases type IH.p.l.c. analysis (van Tilbeurgh et al., 1982) of the

enzymic hydrolysis of the MeUmbLac and MeUmb-(Glc). by CBH I (T. reesei and P. pinophilum) producesactivity patterns as shown in Fig. l(a). Relevant kineticdata (turnover numbers, i.e. catalytic-centre activities)are also indicated. These are only apparent values forthose substrates that yield more than one product. Thesimilarities observed for both CBH I enzymes, isolatedfrom two different organisms, are striking.Only in the case of the first two homologues,

MeUmb(Glc)2 and MeUmb(Glc)3, are specificities asexpected for a cellobiohydrolase. However, with someCBH I samples appreciable hydrolysis at the bondbetween the glucosidic moieties of MeUmb(Glc)2 wasalso observed. This activity was almost completely absentfor enzyme samples further purified by affinity chromato-graphy (van Tilbeurgh et al., 1984), suggesting that acontaminating endocellulose (van Tilbeurgh et al., 1982)or exoglucohydrolase (Wood & McCrae, 1982) waspresent.More complex degradation patterns were obvious for

the higher homologues, MeUmb(Glc).. Thus fromthe tetraoside MeUmb, MeUmbGlc and MeUmb(Glc)2were released in early attack with a linear formation rateof the first two reaction products. Initial reaction veloci-ties were independent and linear in the substrate con-centration range tested (10-2000 ,tM). The inhibitoryeffects of cellobiose and lactose on each reaction productformation were similar, which is preliminary evidence fora single active site.

Hydrolysis of MeUmb(Glc)5 is as complex as thehydrolysis of the tetraoside. The release of each reactionproduct was linear from the start, and no substrateconcentration (20-300 ,M)-dependency of the reactionvelocities could be demonstrated. The MeUmb releasedis probably a secondary reaction product of theMeUmb(Glc)2 hydrolysis.

Deviant specificity could also be demonstrated withcello-oligosaccharides such as cellotetraose, whichyielded significant amounts of glucose (glucose oxidase/peroxidase reagent). As the position of the cleaved bondcannot be unambiguously assigned by using normalcello-oligosaccharides, reducing-end-labelled derivativeswere used. This is illustrated here for the cellopentaose(Fig. 2). The positional and kinetic similarities in the

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(a) A B C D E F A B C D E

I 16(*)

0.9 (0.9)

114(5) l

10.8 (*)t0.8 t0.3(*)

t* (*) 1 (3 2 44 (1.2)(3.2)

(b) A B C D E F

I0.8(0.1)

l 9(17)

117 (7) 121 (6)

l 0.8 (*)

*(*) 17.4 (*)

1*(*) 1*(*) '13.3(*)

A B C D E

10.8(*)

1*(*) 1*(*)Fig. 1. Degradation patterns of cello-oligosaccharides and their chromophoric glycosides by (a) CBH I and (b) CBH II from T. reesei

and P. pinophilum

Mixtures of substrates (30-500 /LM) and CBH I (0.1-1 /tM) or CBH II (0.05-0.5S/M) were incubated (at pH 5.0 at 25 'C) andanalysed (by h.p.l.c.). Arrows indicate bonds hydrolysed as deduced from the chromophoric or radioactive reaction productsobserved. The numbers corresponding to the turnover values (min-') were calculated from the linear rates of the reactionsmonitored (5-30 min) as described in the Experimental section. The values in parentheses are those found for the P. pinophilumenzymes (*, turnover numbers not determined). Symbols: A, f,-(1 --4)-galactopyranosyl; El, ,-(1 --4)-glucopyranosyl; 0,reducing-end glucopyranosyl; 0, 4-methylumbelliferyl group. Letters A-F refer to the putative binding sites on the CBH I.

100

i)00

50

A0Cu--.021

0 20 40 60Time (min)

'ig. 2. Time course of hydrolysis of I1-3Hlcellopentaose byCBH I from T. reesei

[1-3H]Cellopentaose (5 mM; 0.1 GBq/mol) (at pH 5.0 at25 °C) was incubated with CBH I (0.1 ,uM), and samples(1O u1) were removed at times given and diluted (1:4)in acetonitrile/water (4:1, v/v) containing a mixtureof unlabelled cello-oligosaccharides (degrees of poly-merization 1-5, 1 mm each) as reference. After injection(20,1) on to the h.p.l.c. column (see the Experimentalsection), fractions (approx. 0.05-0.5 ml) corresponding toeach oligosaccharide (refractive-index detection) were col-lected and their radioactivities were determined (by liquid-scintillation counting).

degradation of the cellodextrins compared with theirchromophoric derivatives provide evidence for equiva-lencies in binding of glucosyl moieties, as shown (Fig.la). The phenolic residue in MeUmb(Glc)2 then seems tooccupy subsite C, which is, in the case of cellotriose, filledby the reducing-end glucosyl residue.With MeUmbLac and MeUmb(Glc)2 as substrates,

normal Michaelis-Menten kinetics for the release ofMeUmb were found (Table 1). The Km value of thelactoside was about 10 times higher than that for thecellobioside. The catalytic efficiencies of the enzyme forboth types of substrates were nearly identical; hydrolysisof cellotriose was, however, much less efficient.The 2'-chloro-4'-nitrophenyl derivatives are useful

substrates for continuous assays, and the kinetic para-meters for the lactoside are given in Table 1. Thesecompounds are also hydrolysed by some endoglucanasesisolated from T. reesei (Claeyssens, 1988) and P. pino-philum (K. Bhat, T. M. Wood & M. Claeyssens, un-published work), and for comparative purposes thekinetic parameters for endoglucanase I from T. reesei areincluded.

Specificity of cellobiohydrolases type IIThe specificity of CBH II from T. reesei for the

hydrolysis of MeUmb glycosides derived from the cello-oligosaccharides was described in a previous study (van

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Tilbeurgh et al., 1985). The data presented here (Fig. lb)update these results and compare the specificities of thetwo fungal type II cellobiohydrolases. The formation ofMeUmb was never observed, and the binding schemeproposed seems to be valid for both enzymes, namelythat three contiguous sites occupied by glucosyl moietiesare a prerequisite for hydrolysis at the bond locatedpenultimate to the non-reducing end.

For the unmodified cellotriose and cellopentaose thebonds cleaved were determined by using the sugarsradioactively labelled at the reducing end, as reportedabove for CBH I. Turnover numbers for MeUmb(Glc)3and cellotriose were identical, proving that the presenceof the MeUmb group does not appear to influence theoverall reaction rate. In contrast with CBH I, theinfluence of the aglycon on the association constantsis, however, important (Table 2), indicating that thearomatic group is strongly implicated in the interaction ofthe chromophoric ligands and substrates with the CBHII. The concomitant quenching of the fluorescence wasused to determine the association constants and thermo-dynamic binding parameters of some of these ligands(van Tilbeurgh et al., 1985).

Table 1. Comparison of kinetic constants for some substrates ofCBH I and EG I from T. reesei

Activity measurements were performed as described in theExperimental section (at pH 5.0 at 25 °C). Abbreviation:N.D., not determined.

CBH I EG I

Km kcat./Km Km kcat./KmSubstrate (uM) (min-'' M-1) (mM) (min-' * M-1)

MeUmbLacMeUmb(Glc)2CNPhLac*Cellotrioset

26020

504184

6.1 x 1044.5 x 1042.2x 1044.3 x 103

1.41.8

N.D.2.4

3.2x 1053.0x 105N.D.

2.3 x 105* At pH 5.7 (at 25 °C).t Glucose determined.

0.008

0.006

0

-0.004

-0.008

(a)

Wavelength (nm)

(b)

300 325 350 375 400Wavelength (nm)

Fig. 3. Ligand difference absorption spectra for N2PhSC andN2PhSLac binding to CBH I from T. reesei

Spectra were measured (at 15.8 IC) in tandem mixingcuvettes (Yankeelov, 1963). (a) 39 #M-N2PhSC and 50/M-CBH I: spectrum a, baseline (before mixing); spectrum b,spectrum after mixing of sample cuvette; spectrum c, aftermixing of both sample and reference cuvettes; spectrum d,after adding saturating amounts of cellobiose (0.1 M) to thereference cuvette. (b) 32 ,uM-N2PhSLac and 50 ,tM-CBH I:spectrum a, baseline; spectrum b, after mixing of samplecuvette; spectrum c after mixing sample and referencecuvettes; spectrum d, after the addition of cellobiose(0.1 M) to the reference cuvette.

Table 2. Thermodynamic parameters for binding of some chromophoric and non-chromophoric ligands on to CBH I and CBH II fromT. reesei (at pH 5.0)

Association constants were determined either by adiafiltration (Claeyssens et al., 1985), bligand difference spectrophotometry,cprotein difference spectrophotometry, dligand fluorescence quenching titrations (van Tilbeurgh et al., 1985) or edisplacementtitrations of ligand-fluorescence quenching (van Tilbeurgh et al., 1985).

K (at 25 °C) (M-1)AGO (at 25 °C)

(kJ mol-1)AH0

(kJ mol-1)ASO

(J mol- -K-1)

(a) CBH IN2PhS(GlC)2CellobioseCellotrioseLactose

(b) CBH IIMeUmb(Glc)2CellobioseCellotriose

(6.1 +0.4) x IO0ab(5.4 +0.7) x104c(8.0 +0.2) x 104c(3.2+0.1)x 103c

(2.00 +0.03) x IO0d(5.40 + 0.05) x 102e(6.2+0.1) x 104

Ligand

-27.0+0.1-26.7 +0.5-27.9+0.4-20.0+0.6

-30.40 + 0.05-15.60+0.04-27.40+0.04

-58 + 1-47 + 8-33 +9-53 + 6

- 11.6+ 1.7-28.3 +3.3-34+ 2

- 103 +4-68 + 28- 17+ 12- 110+ 55

63 + 6-42 + 13-24+ 7

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x7

10

5

0

Wavelength (nm)

0.006

0.004

1 2 3103 x AA

Fig. 5. Titration of the difference absorption spectrum of CBH Iand cellobiose

Conditions were as described in the Experimental section.Both cuvettes contained 2.141 ml of CBH I solution(12.4,UM), and 0-125 ,ul of cellobiose (5 mM) or 0-125 ,1of sucrose (5 mM) was added to the sample and referencecuvette respectively (at pH 5.0 at 29.4 °C). The sum of thechanges in absorption at 290 nm and 294 nm (A) and thefree ligand concentration ([L]) were used in the calculations(De Boeck et al., 1984). The linearized titration curveis shown. From the slope K= (2.7+0.1)x l04 M-1 iscomputed. The intercept on the AA axis corresponds toAAmax = (4.67 + 0.2) x 1 -3.

0.002

0

Ib

290 300Wavelength (nm)

310 320

Fig. 4. Protein difference spectrum for the binding of cellobiose(a) and lactose (b) to CBH I from T. reesei

Conditions were as indicated in Fig. 3 legend. (a) Spectruma, baseline recorded with 0.72 mM-cellobiose in the samplecuvette and 0.72 mM-sucrose in the reference cuvette;spectrum b, difference spectrum recorded after the additionof CBH I (9.3 /tM) to both cuvettes. (b) Spectrum a,baseline recorded with 7.2 mM-lactose in the sample cuvetteand 7.2 mM-sucrose in the reference cuvette; spectrum b,difference spectrum recorded after the addition of CBH I(9.3 /tM) to both cuvettes.

Thermodynamics of the binding of small ligandsAs in the case of CBH II (van Tilbeurgh et al., 1985),

either chromophoric ligands were tested as indicatorligands in binding studies ofCBH I, or unmodified cello-oligosaccharides were used as perturbants of the proteinspectrum.The determination of the number of sites of interaction

for chromophoric ligands such as 2',4'-dinitrophenyl1-thio-D-cellobioside was carried out by forced-flowdiafiltration, as described for the parent lactoside

(van Tilbeurgh et al., 1982): an association constantK=6.1x104+0.4x104M-1 (at 25°C) for 1.2+0.05binding sites was obtained. These results correspondto the findings by Aluralde & Ellenrieder (1985), whoreport a single binding site for cellobiose on CBH I(K = 2.3 x 104 M-1, at 30 C).

U.v.-absorption spectra of the same chromophoriccompounds show distinct shifts on binding (Fig. 3),which could suggest a different micro-environment forthe two ligands (cellobioside and lactoside). Both differ-ence spectra, however, disappear after the addition ofeither cellobiose or lactose.

Binding of non-chromophoric ligands to CBH I pro-duces very similar protein difference spectra (Fig. 4). Inall cases maxima were observed at 285 nm and 290 nmand a minimum at 294 nm.

Association constants for cellobiose were obtained bytitration of the CBH I difference spectra at 290 and294 nm (Fig. 5); cellotriose binding was measured at291 nm (results not shown). Hydrolysis of this ligandcould be neglected under the conditions used. Thetemperature-dependence of these constants for bothligands is shown in Fig. 6. Values include associationconstants found by kinetic measurements [cellobiose ascompetitive inhibitor ofMeUmb(Glc)2] or by equilibriumbinding experiments as described here or as reported inthe literature (Aluralde & Ellenrieder, 1985). Thermo-dynamic parameters for these cello-oligosaccharidesand lactose are listed in Table 2. Binding parameterswere determined for the latter ligand by titration ofdifference spectra at 285 nm and 291 nm (results notshown). Titrations of cellotetraose and cellopentaose didnot allow linearization according to a simple bindingmodel, probably owing to complicating hydrolysis re-actions. However, titrations at lower temperature indi-cate tentatively that the association constants do notappreciably increase as a function of the chain length inthis oligosaccharide series.

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M. Claeyssens and others

12

4C 1 1

C 11

(a)

A2

3.3 3.4 3.5103/T (K-1)

(b)

*z/

10 L_3.3 3.4 3.5

103/T (K-1)

Fig. 6. van't Hoff plots for the binding of (a) cellobios% and (b)cellotriose to CBH I from T. reesei

For (a) the values of K were obtained by (1) proteindifference absorption titrations as in Fig. 5 (@) and (2)steady-state measurements with cellobiose as competitiveinhibitor of the hydrolysis of MeUmb(Glc)2 or CNPhLac(A). For (b) the K values were obtained by proteindifference absorption titrations (not shown).

DISCUSSIONThe chromophoric derivatives of the cello-oligo-

saccharides and lactose are sensitive tools in the com-parative studies of the cellobiohydrolases and endo-cellulases of fungal and bacterial origin (Claeyssens,1988). In the present study a clear differentiation betweenthe CBH I and CBH II enzymes, isolated from eitherT. reesei or P. pinophilum, was obtained with thesechromophoric substrates (Figs. la and lb).The specificities of the CBH I enzymes are identical for

both organisms and the kinetic constants are very similar.The same conclusion is valid for CBH II enzymes. Thusboth types of CBH seem to be functionally conserved,and this is supported by strong evidence resulting fromstructural studies on the enzymes from both sources (M.Claeyssens, unpublished work).As demonstrated in previous work (Tomme et al.,

1988), CBH I and CBH II (T. reesei) are both stronglyadsorbed on insoluble cellulose, but from the presentstudy important differences in substrate specificities be-come evident. As also reported before (van Tilbeurghet al., 1985), the specificity of the CBH II is more strict,

since the presence of three or four contiguous ,l-(1-+4)-linked glucosyl residues is required and no cleavage atthe heterosidic bonds is observed. The use of reducingend-labelled (radioactive) cello-oligosaccharide made itpossible to establish that a similarity existed in thedegradation pattern of chromophoric substrates (Fig.lb). If the binding model presented in our earlier study(van Tilbeurgh et al., 1985) is still valid, subsites A, B andC are specific for glucosyl residues, whereas the aglyconegroup binds in subsite D [e.g. for MeUmb(Glc)3]. Thehydrolytic cleavage site is situated between subsites Band C. Subsite C can also accommodate a chromophoricgroup, but in this case no hydrolysis occurs [e.g. forMeUmb(Glc)2]. The apparent kinetic parameters showlow and almost constant values for Km and increasingturnover numbers for substrates of higher degree ofpolymerization. The absolute values exceed those foundin the case ofCBH I, and in this respect CBH II is similarto some endocellulases. Thus the mode of action oninsoluble substrates differs from action on solublesubstrates.The CBH I enzymes hydrolyse both cellobiosides and

lactosides and attack at sites other than the non-reducing-end cellobiosyl residues in the higher homologues[MeUmb(Glc)., n > 3]. That this is not an artifact (dueto the presence of chromophoric groups) is proven by thefact that the unsubstituted cello-oligosaccharides showsimilar degradation patterns and kinetic parameters(Fig. la).The influence of the chain length on the kinetic

parameters of the substrates shows no definite trend. Asthe Km values are experimentally not measurable(<10/IM) the apparent turnover numbers are indicativeof the catalytic efficiency. In all cases where they weredetermined it appears that these numbers are very lowwhen compared with other enzymes such as endo-glucanase, e.g. EG I from T. reesei (Table 1). EG I andCBH I from T. reesei show a strong amino acid sequencehomology (Bhikhabhai & Pettersson, 1984) and share,together with other components from the cellulase com-plex, a conserved C-terminal peptide (Knowles et al.,1988). The data presented here suggest that the enzymesalso resemble each other in specificity, and thus probablythe homology extends to the active site of the enzymes.In its action on native cellulose, however, CBH I operatesin a different manner from EG I, and this is almostcertainly due to the presence of the C-terminal peptide,which functions as a substrate-recognition domain inCBH I (Tomme et al., 1988), but whose function in EGI has still to be established.The active-site topology of CBH I was further studied

by equilibrium binding experiments. The data suggestthat a single binding site exists for lactosides and cello-biosides, and this was supported by the identical proteindifference spectra obtained with lactose and cellobiose(Fig. 4) respectively. This specific binding site is spatiallyrestricted, as indicated by the apparent independency ofthe thermodynamic constants from the degree of poly-merization of the cello-oligosaccharides. It is probable,however, that lactosides bind in a slightly different con-formation, since specific parameters (Table 1) and distinctligand difference spectra (Fig. 3) were found for 1-thioanalogues of these substrates.The interaction constants and thermodynamic para-

meters for the binding of some ligands common to CBHI and CBH II (T. reesei) are compared in Table 2.

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Different active-site architecture and/or interactionmodes can be deduced from these data, corroboratingthe conclusions reached above.

For the binding of lactose to CBH I the entropycontribution dramatically decreases compared with cello-biose and cellotriose. This could indicate different con-formations of the enzyme-ligand complexes, leading toa less stable intermediate for lactosides when bound insubsites ABC.The high interaction energy of chromophoric cello-

biosides to CBH II is particularly dominated by itsentropy term. The binding mode in the non-productivecomplex must be totally different from that of a truesubstrate (e.g. cellotriose).The binding constants of cellobiose differ widely for

the two cellobiohydrolases, whereas those from cello-triose are comparable. The effects of this reaction producton the hydrolysis of native cellulose are typical and haveindeed been shown to differ from both enzymes (vanTilbeurgh et al., 1988).

In conclusion, it is clear that the specificities of CBH Iand CBH II from both Trichoderma and Penicillium,classified as exocellobiohydrolases, can be better definedby the use of low-molecular-mass substrates and ligands.With the use of these substrates some information con-cerning the reaction mechanisms of the enzymes and thebinding modes for small ligands emerged, but no answerto the question of the nature of the synergistic action ofthe enzymes on native cellulose was found. Thereforesome hypotheses put forward (Wood & McCrae, 1986)may still be valid. Further work is required to establishmore detailed data on the adsorption characteristics ofthese enzymes and the reaction mechanisms involved insolubilizing microcrystalline cellulose.

M. C. is indebted to N.F.W.O. (Belgium) for grants. P. T. isan I.W.O.N.L. bursary (Belgium). M.C. and T.M.W. thankN.A.T.O. for travel grants. The gift of labelled cello-oligosaccharides by Dr. P. Biely (Bratislava) is gratefullyacknowledged.

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Received 18 January 1989/3 March 1989; accepted 6 March 1989

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