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Structure and function of a cellulase from Penicillium notatum as studied by chemical modification and solvent accessibility
ARCHIVES
OF
BIOCHEMISTRY
Structure Studied
and
AND
126, 77&i-784 (1968)
BIOPHYSICS
Function
of a Cellulase
by Chemical
Modification GtiRAN
Inslitzcte
of Biochewktry, Received
May
from and
Penicillium
Solvent
notatum
as
Accessibility
PETTERSSON
University
of Uppsala,
25, 1968; accepted
Uppsala,
Sweden
June 7, 1968
A cellulolytic enzyme from Penicillium notatum has been studied by chemical modification, spectrophotometric and spectrofluorometric methods. Two to five tryptophyl residues out of 13 behave as if they were accessible. The oxidation of two tryptophyl residues causes a complete loss in enzymic activity. Perturbation spectrophotometry using cellobiose (a product of the enzyme reaction) as neutral pertrlrbant indicates a specific interaction between cellobiose and trypt.ophyl residues. The pH profile of the difference peak at 292 nm shows a striking resemblance with the pH activity curve. The inactivation of the enzyme by coupling with diazonium-1-H-/tetrazole (DHT) and the shape of the pH activity curve indicate that histidyl residues are involved in the catalytic mechanism. Five tyrosyl residues can be nitrated in the native enzyme and the activity decreases concomitant to 35% of the initial value. In the heat denatured enzyme all 14 tyrosyl residues can be nitrated. Succinylation of 60% of the amino groups does not affect the activity.
A cellulase from Penicillium notatum has been purified and characterized (1, 2). The enzyme consists of one polypeptide chain crosslinked with one disulfide bridge. The molecular weight was determined to be 35,000. Amino acid analysis revealed a high content of acidic and aromatic amino acids. The enzymatic stability was also investigated together with a physico-chemical study of the interaction with hydrogen ions (3). The enzyme was found to be sta,ble in the region pH 4-8.5 at room temperature. The decrease in stability on the alkaline side occurred concomitantly with the titration of c-amino groups. Among the inhibitors studied mercuric ion was found to be very eficient. Difference spectrophotometric measurements indicated that this inhibition could possibly be ascribed to interaction with tryptophyl residues (4). At present little is known about the relation between structure and function of cellulolytic enzymes, since few cellulases have been isolated and characterized (5). However, lysozyme, which is functionally related to the cellulases (both attack p-1,4-
glucosidic bonds) is one of the most thoroughly studied enzymes so far (6). It has been shown for lysozyme that tryptophyl residues are involved in the binding of the substrate to the enzyme molecule (7, 8). These results together with our previous findings which indicate that tryptophyl residues may have a similar function in the cellulolytic enzymes (4) prompted further studies on the struct.ural and functional properties of these residues. To get a more complete picture of the structural and functional properties of the enzyme the reactivity of the tyrosyl, histidyl, and amino groups have also been examined. MATERIALS
AND
METHODS
Ultraviolet absorbance was measured with a Zeiss PM& II spectrophotometer, when whole spectra were required a Beckman DB recording spectrophotometer was used. The absorbance maxima were checked with the Zeiss spectrophotometer. Fluorescence intensities were measured with a Turner spectrofluorometer Model 210 “Spectra.” Cellulase activity was routinely determined from 776
STRUCTURE
AND
FUNCTION
the reducing sugars formed after incubation of the enzyme with carboxymethylcellulose. (9). A spinco Model 120 B automatic amino acid analyzer was used for the amino acid analyses. The pH optimum of the enzyme was determined using hydroxyethylcellulosel as substrate. 0.4y0 (w/v) substrate solutions were prepared in the following buffer systems: pH 2.4-3.2 glycine-HCl, pH 3.G-5.2 NaAc-HAc, pH 6.0-7.5 sodium phosphate, pH 7.9-8.6 Tris-HCI. The ionic strength of the buffers was 0.05. In this case the cellulase activity was determined visocometrically using a 5901 B Auto Viscometer from Hewlett Packard. The efflux time of the viscometer, which had an efflux time for water of 15 set, was determined every minute a,fter the mixing of the enzyme solution (100 ~1, Apa0 = 0.116) with the substrate (10 ml). Measurements were made during the first 15 min of the reaction. The temperature was 25”. The data werf: treated according to Almin and Eriksson (10) so that the arbitrary enzymic act,ivity B could be estimated at each pH. It should be observed that the value of a, the empirical constant used for the estimation of B, was 1.5. A much higher value was obtained by Almin and Eriksson (IO) using CMC as substrate. The inhibition with riboflavin-5’.phosphate and histamine was studied in the following way. Solutions of the inhibitor were prepared in 0.2 M sodium acetate buffer pH 5.0. A small aliyuot of enzyme solution was added, until the absorbance at 280 nm was approximately 0.05, and the solutions were left at room temperature for 30 min. The activity was then tested against a CMC solution which contained inhibitor of the same concentration as in the inhibitor solution. Tryptophyl residues were modified by oxidation with Wbromosuccinimide (NBS) according to Spande and Witkop (11) and by reaction with 2hydroxy-5-nitrobenzylbromide according to Horton and Koshland (12). The iV-bronnosuccinimide reaction was carried out in 0.1 M sosdium acetate buffer pH 5.0. To 5 ml of 0.027, solution of cellulase 10 ~1 aliquots of 10-a M NBS were added under magnetic stirring. After each addit’ion of NBS the absorbance at 280 nm was determined and 20 ~1 of the solution were withdrawn and tested for cellulase activity. It was found that vigorous stirring was necessary to avoid local excess of reagent, which otherwise caused turbidity in the solution. A similar experiment was carried out in 8 M urea. The number of tryptophyl residues oxidized was calculated from the empirical relationship (11) 1 Obtained from MO och Domsjij AB, Ornskiildsvik, Sweden. Batch 4113. Molecular degree of substitution (MS) = 0.51.
777
OF A CELLULASE
n=
1.31’A&& 5500 X molarity
cellulase
’
where AAzso is the maximum difference in absorbance. Reaction of the tryptophyl residues with 2-hydroxy-5-nitrobenzylbromide (Z Br) was performed as described by Horton and Koshland (12). To 2 ml samples of cellulase (A280 = 2) in 0.1 M sodium acetate buffer pH 5.0, aliquots of # Br dissolved in dried aceton were added to a final molar ratio of 2O:l (6 Br:tryptophan). A small aliquot of the sample was withdrawn and tested for enzymic activity. The reaction mixture which contained some precipitate was then dialyzed for 2 hr against 0.1 M glycine-NaOH buffer pH 10.5, 8 M urea. The solution, which now was free from precipitate was run on a Sephadex G25 column 1.5 X 20 cm in the same buffer. The protein fractions were collected and the number of 2-hydroxy-5-nitrobenzyl groups covalently bound was estimated from the absorbance at 410 nm assuming a molar extinction coefficient of 18,000 (12). Nitration of tyrosyl residues was carried out using tetranitromethane TNM as described by Sokolovski et al. (13). A!iquots of a 0.84 M solution of TNM in 95y0 ethanol were added to l-ml samples of protein. One series of samples contained native enzyme while the other contained heat denatured enzyme obtained by incubation at 80” for 15 hr. The reaction mixture was left for 4 hr at room temperature (22’) and was then gel filtered on a Sephadex G-25 column (1 X 15 cm), in 0.05 M Tris-HCl buffer pH 8.0. The protein fractions were pooled and tested for enzyme activity. The extent of modification was estimated by measuring the absorbance at 428 nm assuming a molar extinction coefficient of 4100 (13) for 3-nitrotyrosine. In another experiment, samples of native and heat denatured cellulase were modified according to the procedure described using a molar excess of reagent (TNM: tyrosyl residues) of 80. After modification the protein was hydrolyzed for 24 hr with 6 M HCl at 110” and subjected to amino acid analysis. Coupling with diazonium-1-H-tetrazole (DHT) was carried out as described by Horinishi et al. (14) and as modified by Sokolovsky and Vallee (15). DHT was prepared from amino-1-H-tetrazole (14). The concentration was determined by measuring the formation of the monoazoderivative of N-acetyltyrosine (15). The coupling of DHT with the enzyme was performed in 0.67 M NaHC03 pH 8.8 at room temperature. The extent of reaction was followed by measuring the absorbances at 480 nm (biazohistidine) and 550 nm Activity measurements were (biazotyrosine). carried out along with the coupling reactions.
778
PETTERSSON
Succinylation was performed by adding solid succinic anhydride stepwise to a solution of cellulase in 0.2 M potassium phosphate buffer pH 7.5. The protein concentration was usually 1 mg per ml. After each addition of succinic anhydride the pH was checked and if necessary adjusted to the original value with 0.1 M NaOH. The final molar excess (succinic anhydride:amino groups) was 100. The extent of succinylation was determined with the ninhydrin method according to Moore and Stein (16) using phenylalanine as a standard. To study the apparent accessibility of tryptophan groups the neutral solvent perturbation technique by Herskovits and Laskowski (17) was applied. Sucrose and cellobiose were used as perturbants. Two well-matched l-cm tandem silica cells were used. The protein concentration was approximately 0.5 mg per ml. Spectra were recorded at the following pH values, 2.1, 2.9, 4.1, 5.0, 6.0, 7.1, and 8.1. Using the following buffer systems: HCl-KCl, glycine-HCl, NaAc-HAc, Tris-HCl and glycine-NaOH. The ionic strength was 0.05. The dependence of the intensity of tryptophan fluorescence upon the level of perturbant (18) was determined using propylene glycol as neutral perturbant. Native cellulase was compared with a peptic digest. The peptic digestion was carried out as follows: To 1 ml of cellulase dissolved in 0.01 M HCl, an aliquot of pepsin in 0.01 M HCl was added so that the molar ratio of pepsin:cellulase became 1:lOO. The mixture was left for 15 hr at 37”. The reaction was interrupted by adjusting the pH to 8 with sodium hydroxide. The wavelength of excitation was 290 nm and the wavelength of maximal emission was near 350 nm. Materials. The cellulase was purified as described in (1). 2-Hydroxy-5-nitrobeneyl bromide was obtained from Nutritional Biochemical Corporation, Cleveland, Ohio, USA. Teranitromethane was obtained from Fluka AG, Switeerland and N-acetyl tryptophanamide from Mann Research Laboratories, New York, N.Y., USA.
0x1~~4~10~ OF TRYPTOPHYL WITH NBS
RESIDUES
Oxidation of tryptophyl residues with KBS has been shown to be a specific and convenient method for determining the tryptophan content in proteins (11). The method is based upon t#hedecrease in absorbance at 280 nm accompanying the transformation of the indole to the oxindole chromophere. ii titration of the tryptophyl residues of native cellulase at pH 5 is shown in Fig. 2 (open circles). The initial linear decrease in absorbance with increasing concentration of NBS is followed by a region with nearly constant absorbance. This region actually fol-
2
4
6
6
PH FIG. 1. Effect of pH on the activity of a cellulase from Penicilliunz not&urn. Viscometric deSubstrate: 0.4yo hydroxyethylceltermination. lulose. Enzyme concentration Azgo = 1 X 10-a. Temperature 25”.
RESULTS
DETERMINATION OF THE PH-OPTIMUM OF THE ENZYME
Due to its higher precision the viscometric assay method was chosen in this case. To avoid complications due to protolysis of the substrate hydroxyethylcellulose was used instead of carboxymethylcellulose. The relative cellulase activity B (10) as a function of pH is given in Fig. 1. The curve has a broad maximum in the region pH 4.57 and falls rapidly on both sides of this optimum.
FIG. 2. Oxidation of the tryptophans of cellulase with 0.001 M NBS. Open circles: represent the ratio of decrease in absorbance at 280 nm to the initial absorbance. Solid circles : percent inactivation. Concentration of cellulase: 0.25 mg/ml. Medium: 0.1 M HAc-NaAc pH 5.0. Room temperature (22”).
STRUCTURE
AND FUNCTION
779
OF A CELLULASE
denaturing medium (8 N urea) only 2.7 residues were modified. Activity measurements on the modified product revealed that 60% of the initial activity was recovered. ACCESSIBILITY
10 NBS/cellulase
20 mole/mole
FIG. 3. Oxidation of the tryptophans of cellulase with 0.01 M NBS in 8 M urea. Open circles: the ratio of decrease in absorbance at 280 nm to the initial absorbance. Concentration of cellulase: 0.25 mg/ml. Medium: 0.1 M HAc-NaAc, 8 M urea. Room temperature (22”).
lows by one with a slow increase in absorbance due to opalescence. The plateau value corresponds to the titration of 2.3 tryptophan groups. From Fig. 2 it may also be seen that the activity (filled circles) decreases concomitantly with the oxidation of tryptophyl residues, and that the plateau value for complete inactivation roughly coincides with the plateau value for the titration curve. If the titration with NBS is carried out in a denaturing medium (8 M urea) almost all the tryptophyl residues are titrated (Fig. 3). The total decrease in absorbance corresponds to the oxida,tion of 12 tryptophyl residues. The total tryptophan content of the cellulase has previously been determined to be 13 residues per mole. SUBSTITUTION WITH
OF TRYPTOPHYL
OF TRYPTOPHYL
RESIDUES
The neutral solvent perturbation technique of Herskovits and Laskowski (17) was used to examine the perturbation effects of cellobiose, which is a product of the enzyme action and probably an inhibitor, and sucrose which has no inhibitory effect. Perturbation spectra were recorded at pH values from 2.1 to 8.1. A suboptimal concentration of perturbant (10%) was used due to the limited solubility of cellobiose. The perturbation spectrum of cellulase with 10 % cellobiose at pH 4.1 is shown in Fig. 4 (open circles). The main optimum at 292 nm arises entirely from the perturbation of the typtophan spectrum while tyrosyl groups may ha,ve an influence on the peak at 285 nm. The perturbation effect obtained with 10% sucrose under the same conditions is much lower (Fig. 4, filled circles) but the optima appear at approximately the same wavelengths. A control experiment with N-acetyl-n-tryptophanamide showed no significant difference in perturbation effect between cellobiose and sucrose. The perturbation spectra had the same general features over the pH interval studied. The pH dependence of AEM at 292 nm is shown in Fig. 5 with cellobiose (open circles) and sucrose (filled circles) as perturbants. The cellobiose curve has a maximum in the region pH 4-6 while the sucrose curve has its minimum in the pH region 3-5.
RESIDUES
2-HYDROXY-5-NITROBENZYLBROMIDE
2-Hydroxy-5-nitrobenzylbromide p Br is a reagent with a high degree of specificity towards tryptophyl residues in acid and neutral media (12). The only other amino acid that reacts is cysteine (12). With a final molar ratio of 2O:l (9 Br: tryptophan) the extent of modification was determined to be 4.6 residues per mole cellulase. The extent of modification was constant even if the molar excess of reagent was increased to 50: 1. When the reaction was carried out in a
270
280
290
304
A nm
FIG. 4. Perturbation spectra of cellulase. Media: 10% cellobiose, open circles and 10% sucrose, solid circles. Protein concentration: 0.5 mg/ml, pH 4.1.
780
PETTERSSON
2.0 0
0
‘: q.2 w lu
a
0.4
D
PH FIG. 5. The pH profile of the height of the perturbation peak at 292 nm (Aen&. Media: 10% cellobiose open circles, and 10% sucrose solid circles. Protein concentration: 0.5 mg/ml.
D f 8 : :: t 20 0 3 E : .z 30 ‘$ 10 or”
E Percent
10 20 propylene glycol
30
FIG. 6. The intensity of tryptophan fluorescence at 350 nm as a function of concentration of propylene glycol. Open circles: native cellulase, solid circles: a peptic digest of the cellulase. Excitation wavelength 290 nm. pH 5.0. Protein concentration: 0.03 mg/ml.
Fluorescence studies. The accessibility of tryptophyl residues to neutral solvent perturbants has been studied by Steiner et al. (18) by measuring the dependence of fluorescence upon the concentration of perturbant. This method was adopted to examine the accessibility of the tryptophyl residues of the cellulase. As neutral solvent perturbant propylene glycol was used and as standard a peptic digest. In Fig. 6 the relative Auorescence intensity is plotted as a function of the concentration of propylene glycol. The intensity of fluorescence of the cellulase increases linearly with the concentration of propylene glycol and the slope is 29 % of that of the peptic digest. The figure 29% means that 3.8 tryptophans out of 13 behave as if they were exposed to the perturbant. This figure may however have a very limited
quantitative significance owing to the fact that different tryptophyl residues can have different quantum yields. The method described above was also used to check the perturbation effect of cellobiose and sucrose. Though these experiments have not yet been terminated it is evident that there is no significant difference between cellobiose and sucrose as perturbants in this case. Nitration of tyrosyl groups. Tetranitromethane (TNM) has been shown to be a mild and specific reagent for the nitration of tyrosyl groups in proteins (13). If increasing amounts of TNM are added to native cellulase the nitration of tyrosyl groups increases until a plateau is reached (see Fig. 7, open circles). The plateau value corresponds to the nitration of 4 to 5 tyrosyl groups. The enzymic activity decreases concomitant with the nitration of tyrosyl groups (Fig. 7, squares) and reaches a plateau region at about 35 % of the initial activity. At higher concentrations of TNM there seems to be a decrease in activity again. In heat-denatured cellulase, 11-12 tyrosyl residues are nitrated. This plateau level is reached very rapidly as can be seen from Fig. 7 (filled circles). Samples of native and heat-denatured cellulase were nitrated with TNM using a molar excess of 60: 1. The samples were hydrolyzed and subjected to amino acid analysis. A
0
J 20 40 60 60 100 TNM/ tyrosyl residues mole/mole
FIG. 7. Nitration of cellulase with TNM. The formation of 3-nitrotyrosyl residue and the effect on the enzymic activity of increasing amounts of TNM. Open circles: native enzyme. Solid circles: heat denatured enzyme. Squares: Cellulase activity. The reaction was performed at pH 8.0 at room temperature. The degree of nitration was determined spectrophotometrically at 428 nm.
STRUCTURE
AND
FUNCTION
781
OF A CELLULASE
new peak appeared in the chromatogram at the same place as 3nitrotyrosine (13). In the case of denatured enzyme the tyrosine peak almost completely disappeared which indicated that all tyrosyl residues were actually modified. In the case of native cellulase the tyrosine peak was reduced corresponding to the nitration of 5 tyrosyl residues. COUPLING WITH DIAZONIUM-1 II-TETRAZOLE DHT
20 OHT lcellulose
The coupling reagent diazonium-l-Htetrazole DHT was introduced by Horinishi and co-workers (14) for spectrophotometric determination of histidine in proteins. The method has later been modified by Sokolovsky and Vallee (15). Contrary to the other reagents used in this work DHT is very unspecific and apart from histidine and tyrosine it also reacts with amino and hydroxyl groups. Figure 8 shows the absorbances at 480 (biozohistidine, open circles) and 550 nm (biozotyrosine, filled circles) as a function of -the concentration of DHT. The absorbance at 480 nm increases rapidly at first then more slowly. -4 true plateau value is never reached in spite of the very high excess of reagent used. It may also be seen from Fig. 8 that the absorbance at 550 nm also increases almost linearly upon addition of DHT. It is thus evident that biazotyr0sin.e or possibly monoazotyrosine, which also has some absorbtion at 550 nm (15) f orms concomitant with biazohistidine and that it is not possible to determine histi-
0.5
5 5 0.3 G 6 f :: 0.1 9
0.5
E 0.3 % Y) % P 0 s
0.1 2 a
40
60
mole/mole
FIG. 9. The effect of increasing amounts of DHT on the cellulase activity. Coupling condi-
tions: bicarbonate buffer pH 8.8, reaction time 30 min, room temperature, protein concentration 0.2 mg/ml.
dine independently of tyrosine before all histidyl and tryosyl groups have reacted to form the biazoderivatives. The enzyme is rapidly inactivated upon addition of DHT (Fig. 9) and the inactivation is practically complete at a molar excess of 60, (DHT: enzyme) \vhich is before any significant amounts of colored products have been formed. SUCCINYLATION WITH SUCCINIC ANHYDRIDE
Succinic anhydride reacts specificly with amino groups but under specific conditions also with hydroxyl groups (19). If native cellulase is treated with succinic anhydride with a molar excess of approximately 100 (succinic anhydride: amino group) about 60 % of the amino groups are modified. This figure was obtained after determination of the amino groups with ninhydrin and is of course only a rough estimate. It is to be observed that the degree of substitution was roughly independent of the molar excess of reagent in the region 20-200 (succinic anhydride: amino group). The enzymic activity was uneffected or increased somewhat upon succinylation. INHIBITION STUDIES WITH RIBOFLAVIN5'-PHOSPHATE AND HISTAMINE
FIG. 8. Coupling of DHT with cellulase as determined by the absorbance at 480 and 550 nm. Coupling conditions: bicarbonate buffer pH 8.8, reaction time 30 min, room temperature, protein concentration 0.2 mg/ml.
Riboflavin-5’-phosphate has been shown to form red complexes with free indole compounds and with tryptophan bound in proteins (20). Complexes of the charge trans-
782
PETTERSSON
dized with NBS before precipitation occurred, and that 4.6 could be modified with p Br. The fact that only 2.7 residues were modified with $?JBr in the urea-denatured see text.) enzyme seems nevertheless to indicate that some other factsor than difference in exposure activity Cont. of inhibitor (M) Enzyme Inhibition (% of initial) of the tryptophyl residues was limiting in this case. It is to be observed that 12 tryp81 1.0 x 10-z RFP 72 2.0 x 10-z tophyl residues were titrated with NBS if the reaction was carried out in 8 M urea. 74 Histamine 1.0 x 10-t Further evidence for the limited accessibility 68 2.0 x 10-z of the tryptophyl groups have been achieved 51 8.0 X 1O-2 from the spectrofluorometric measurements (see Fig. 6). Oxidation of 2.3 tryptophans fer type have also been identified between with NBS caused complete inactivation of imidazole and indole compounds. Moreover the enzyme. From Fig. 2 it may be seen that imidazole derivatives have been found to be there is a good parallelism between the activinhibitors of lysozyme (21). The results given ity curve and the decrease in absorbance at in Table I show that riboflavin and histamine 280 nm. This may indicate that no other are weak inhibitors of the cellulase activity. amino acid side groups have reacted beside the tryptophyl residues. The low consumpDISCUSSION tion of NBS, 7 moles per mole of proThe pH profile of an enzyme reaction may tein points in the same direction. The be used for identifying the groups on the modification of 4.6 tryptophyl residues with enzyme molecule that are involved in the @ Br caused only a 40 % inactivation. If one catalytic mechanism. Generally a determinasupposes that the same tryptophans have tion of the pH dependence of the Michaelis reacted, which of course is not certain, these parameters V and K, as well as the pH results seem to contradict each other. On the stability is needed to properly exploit the other hand it is reasonable that oxidation possibilities of this method (22). The pH of indole groups to oxindole derivatives may profile of the cellulase may be seen in Fig. 1. cause a greater change in the sturcture This is a composite curve which partly may around the indole group than int,roduction depend on changes in K, and partly on of a benzene ring. It is as a matter of fact changes in V. Though initial velocities have astonishing how sensitive many enzymes are been measured it is not ruled out that the to oxidation with NBS (11) even at a rather fall in activity on the acidic side of the optilow excess of reagent where the risk for s&tmum may be due to an irreversible con- ting peptide bonds is minimal. formation change. It has been previously Cellobiose, which is a product of the found that the stability of the enzyme de- enzyme reaction was tried as a neutral percreases rapidly at pH values below 3 (3). The turbant using the difference spectrophotomfall in activity on the alkaline side might etry method according to Herskowits and depend on a change in the state of ionization Laskowiski (17). It was found that cellobiose of a group with a pK, value of 7.5 (see Fig. gave rise to a typical tryptophan spectrum 1). This might be an amino or more probably and that the perturbation effect was much an imidazole group. The substrate is com- more pronounced than with sucrose as perpletely non-ionic and should thus have no turbant (see Fig. 4). Moreover the pH profile effect on the pH profile. of the difference peak at 292 nm (Fig. 5) The enzyme contains 13 tryptophyl resi- displays a close resemblance with the pH dues of these only a few seem to be exposed. activity curve (Fig. 1). The corresponding This has been found earlier from difference curve for sucrose has quite a different spectrophotometric measurements and has appearance (Fig. 5). It must be emphasized now been further elucidated. It was found that that these results do not necessarily mean approximately 2 tryptophans could be oxi- that there is a specific interaction between TABLE
INHIBITION
I
OF A CELLULASE FROM notatum WITH RIBOFLAVIN-5’-PHOSPHATE AND HISTAMINE (For experimental
Penicillium RFP details
STRUCTURE
AND FUNCTION
cellobiose and tryptophyl residues on the protein. It is also possible that cellobiose can perturb trypt’ophyl residues not accessible to other perturbants by some specific mechanism, without the presence of any direct binding. If the active site of the enzyme is in a cleft as in lysozyme (7) it is possible that cellobiose has the ability to penetrate into this cleft either directly or by inducing a conformation change. Another indication of a possible role of the tryptophyl residues in the enzymatic mechanism can be shown from the inhibition experiments with riboflavin and histamine. These substances have been shown to form complexes of the charge transfer type with tryptophyl residues in proteins (20, 21). Moreover histamine is an inhibitor of l-ysozyme (21). It is seen from Table I that iiboflavin and histamine inhibit the cellulase, though a rather high concentration is required. From spect’rophotometric titrations it was concluded that the tyrosyl groups titrated freely and with the same pK (3). Since conformation change occurs before the titration of the tyrosyl groups (3), this does not rule out the possibility that some tyrosyl groups might be unexposed in the native molecule. The fact that only 5 tyrosines out of 15 were nitrated with TNM seems to support this hypothesis (Fig. 7). On the contrary after heat denaturation 11-12 tyrosines were nitrated as determined with direct spectrophotometry (14). Amino acid analysis on the other hand revealed that all tyrosyl groups were nitrated. The difference between the two methods was probably due to the limited precision in the spectrophotometric method. Nitration of 5 tyrosyl groups was associated with a loss of 65 % of the initial act,ivity. Considering the fact that nitration causes a shift to lower pH values of the pK of tyrosine it is possible that this loss in activity is due to a change in the stability of the enzyme. Coupling with DHT (diazonium-IH-tetrazole) caused :a complete loss of activity (Fig. 9) at a very low excess of reagent. This happened before any biazoderivatives had been formed as judged by measurements of the absorbance at 450 and 550 nm (15). Since DHT is a rather unspecific reagent it is
OF A CELLULASE
783
difficult to draw any conclusions about the reason for the inhibition. Nevertheless histidy1 groups are probably more reactive than other possible groups. The inhibition might thus be due to formation of monazohistidyl derivatives. 60 % of the amino groups could be succinylated with succinic anhydride. This figure was determined from the reduction in ninhydrin color and must be regarded as a rough estimate. Succinylation did not effect the enzymatic activity. Data suitable for a comparison with the present findings are still, with a few exceptions, rather scanty in the literature on cellulolytic enzymes. Generally fungal cellulases seem to have pH optima on the acid side of neutrality (23). Li et al. (24) found a broad pH optimum similar to that described here for a highly purified endoglucanase from Trichoderma viride. Similar pH optima have also been observed for two highly purified cellulases from Stereum sanguinolentum studied by Bjiirndal and Eriksson (25). Inhibition with NBS has been found by Iwasaki et al. (26) studying two cellulases from Trichoderma koningi. The further work on this enzyme will concentrate on the role of tryptophyl and histidy1 groups for the enzyme action. Moreover the kinetics and mode of action on different cellulose substrates will be studied. ACKNOWLEDGMENT I thank my teacher Professor J. Porath for his kind interest throughout the progress of this work and Mrs. Ulla-Britt Fredriksson for skilful technical assistance. The work was supported by a grant from the Hierta-Retzius foundation. REFERENCES 1. PETTERSSON, G., Arch. Biochem. Biophye. 133, 307 (1968). 2. PETTERSSON,G. AND EAKER, D.L., Arch.Biothem. Biophys. 124, 154 (1968). 3. PETTERSSON, G. AND ANDERSSON, L., Arch. Biochem. Biophys. 134,497 (1968). 4. ERIKSSON, K.-E. AND PETTERSSON, G., Arch. Biochem. Biophys. 134, 160 (1968). 5. NORKRANS, B., Adv. Appl. Microbial. 9, 91 (1967). 6. PERUTZ, M. G., Prac. Roy. Sot. (London) Ser B. 16’7, 348 (1967).
7. BL.~KE, C. C. F., JOHNSON, LOUISE N., MUIR, G. A., NORTH, A. C. T., PHILIPS, D. C. AND SARMA V. R., Proc. Roy. Sot. (London) Ser. B. 167, 378 (1967). 8. RUPLEY, J. A., Proc. Roy. Sac. (London) Ser. B. 167, 416 (1967). 9. PETTERSSON, G. .~ND PORATH, J., in “Methods in Enzymology,” VIII, p. 603, New YorkLondon 1966. 10. ALMIN, K. E. END ERIKSSON, K.-E., Biochim. Biophys. Acta 139, 238 (1967). 11. SPANDE, T. F. -YND WITKOP, B., in “Methods in Enzymology,” XI, p. 498, New YorkLondon 1967. 12. HORTON, H. R. AND KOSHLAND, I). E., JR., J. Am. Chem. Sac. 87,1126 (1965). 13. SOKOLOVSKY, M., RIORD~N, J. F. AND VALLICE, B. L., Biochemistry 6, 3582 (1966). 14. HORINISHI, H., HXHIMORI, Y., KURIHaRa, K. AND SHIB.ITA, K., Biochim. Biophys. Acta 86, 477 (1964). 15. SOKOLOVSKY, M. AND VALLEE, B. L., Biochemistry 6, 3574 (1966). 16. MOORE, S. AND STEIN, W. H., J. Biol. Chem. 176, 367 (1948).
17. HEKSKOVITS, T. T. .\ND ~~~~~~~~~~~~ M. JR., J. Biol. Chem. 237, 2481 (1962). 18. STEINER, 11. F., LIPPOLDT, R., EDI~:LHOCH, H. BND FRBTT~LI, V., Biopolymers Symp. 1 p. 355 (1964). 19. TICRMINIELLO, L., BIER, M. AND NORD, F. F., Arch. Biochem. Biophys. 73, 171 (1958). 20. ISENBERG, I. BND SZENT-GY~RGYI, A., Proc. Alat. Acad. Sci. U.S.A. 44, 857 (1958). 21. SHINITZKY, M., KAT~H~LSKI, E., GRISARO, V. AND SH.IRON, N., Arch. Biochem. Biophys. 116, 332 (1966). 22. DIXON, M. racy WEBB, E. C. “Enzymes,” p. 116. Longmans, Green and Co. Ltd. 2nd Ed. London 1964. 23. G.~SCOIGNX, J. A. AND GBSCOIGNE, M. M., “Biological Degradation of Cellulose,” p. 58. Butterworths, London 1960. 24. LI, L. H., FLORI, R. M. AND KING, K. W., Arch. Biochem. Biophys. 111,439 (1965). 25. BJ~RNDAL, H. AND ERIKSSON, K.-E., Arch. Biochem. Biophys. 124, 149 (1968). 26. 1w.4~~1, T., IKEDA, R., H~Y~SHI, K. AND FUN.~TSU, M., J. Biochem. (Japan) 67, 478 (1965).
OF
BIOCHEMISTRY
Structure Studied
and
AND
126, 77&i-784 (1968)
BIOPHYSICS
Function
of a Cellulase
by Chemical
Modification GtiRAN
Inslitzcte
of Biochewktry, Received
May
from and
Penicillium
Solvent
notatum
as
Accessibility
PETTERSSON
University
of Uppsala,
25, 1968; accepted
Uppsala,
Sweden
June 7, 1968
A cellulolytic enzyme from Penicillium notatum has been studied by chemical modification, spectrophotometric and spectrofluorometric methods. Two to five tryptophyl residues out of 13 behave as if they were accessible. The oxidation of two tryptophyl residues causes a complete loss in enzymic activity. Perturbation spectrophotometry using cellobiose (a product of the enzyme reaction) as neutral pertrlrbant indicates a specific interaction between cellobiose and trypt.ophyl residues. The pH profile of the difference peak at 292 nm shows a striking resemblance with the pH activity curve. The inactivation of the enzyme by coupling with diazonium-1-H-/tetrazole (DHT) and the shape of the pH activity curve indicate that histidyl residues are involved in the catalytic mechanism. Five tyrosyl residues can be nitrated in the native enzyme and the activity decreases concomitant to 35% of the initial value. In the heat denatured enzyme all 14 tyrosyl residues can be nitrated. Succinylation of 60% of the amino groups does not affect the activity.
A cellulase from Penicillium notatum has been purified and characterized (1, 2). The enzyme consists of one polypeptide chain crosslinked with one disulfide bridge. The molecular weight was determined to be 35,000. Amino acid analysis revealed a high content of acidic and aromatic amino acids. The enzymatic stability was also investigated together with a physico-chemical study of the interaction with hydrogen ions (3). The enzyme was found to be sta,ble in the region pH 4-8.5 at room temperature. The decrease in stability on the alkaline side occurred concomitantly with the titration of c-amino groups. Among the inhibitors studied mercuric ion was found to be very eficient. Difference spectrophotometric measurements indicated that this inhibition could possibly be ascribed to interaction with tryptophyl residues (4). At present little is known about the relation between structure and function of cellulolytic enzymes, since few cellulases have been isolated and characterized (5). However, lysozyme, which is functionally related to the cellulases (both attack p-1,4-
glucosidic bonds) is one of the most thoroughly studied enzymes so far (6). It has been shown for lysozyme that tryptophyl residues are involved in the binding of the substrate to the enzyme molecule (7, 8). These results together with our previous findings which indicate that tryptophyl residues may have a similar function in the cellulolytic enzymes (4) prompted further studies on the struct.ural and functional properties of these residues. To get a more complete picture of the structural and functional properties of the enzyme the reactivity of the tyrosyl, histidyl, and amino groups have also been examined. MATERIALS
AND
METHODS
Ultraviolet absorbance was measured with a Zeiss PM& II spectrophotometer, when whole spectra were required a Beckman DB recording spectrophotometer was used. The absorbance maxima were checked with the Zeiss spectrophotometer. Fluorescence intensities were measured with a Turner spectrofluorometer Model 210 “Spectra.” Cellulase activity was routinely determined from 776
STRUCTURE
AND
FUNCTION
the reducing sugars formed after incubation of the enzyme with carboxymethylcellulose. (9). A spinco Model 120 B automatic amino acid analyzer was used for the amino acid analyses. The pH optimum of the enzyme was determined using hydroxyethylcellulosel as substrate. 0.4y0 (w/v) substrate solutions were prepared in the following buffer systems: pH 2.4-3.2 glycine-HCl, pH 3.G-5.2 NaAc-HAc, pH 6.0-7.5 sodium phosphate, pH 7.9-8.6 Tris-HCI. The ionic strength of the buffers was 0.05. In this case the cellulase activity was determined visocometrically using a 5901 B Auto Viscometer from Hewlett Packard. The efflux time of the viscometer, which had an efflux time for water of 15 set, was determined every minute a,fter the mixing of the enzyme solution (100 ~1, Apa0 = 0.116) with the substrate (10 ml). Measurements were made during the first 15 min of the reaction. The temperature was 25”. The data werf: treated according to Almin and Eriksson (10) so that the arbitrary enzymic act,ivity B could be estimated at each pH. It should be observed that the value of a, the empirical constant used for the estimation of B, was 1.5. A much higher value was obtained by Almin and Eriksson (IO) using CMC as substrate. The inhibition with riboflavin-5’.phosphate and histamine was studied in the following way. Solutions of the inhibitor were prepared in 0.2 M sodium acetate buffer pH 5.0. A small aliyuot of enzyme solution was added, until the absorbance at 280 nm was approximately 0.05, and the solutions were left at room temperature for 30 min. The activity was then tested against a CMC solution which contained inhibitor of the same concentration as in the inhibitor solution. Tryptophyl residues were modified by oxidation with Wbromosuccinimide (NBS) according to Spande and Witkop (11) and by reaction with 2hydroxy-5-nitrobenzylbromide according to Horton and Koshland (12). The iV-bronnosuccinimide reaction was carried out in 0.1 M sosdium acetate buffer pH 5.0. To 5 ml of 0.027, solution of cellulase 10 ~1 aliquots of 10-a M NBS were added under magnetic stirring. After each addit’ion of NBS the absorbance at 280 nm was determined and 20 ~1 of the solution were withdrawn and tested for cellulase activity. It was found that vigorous stirring was necessary to avoid local excess of reagent, which otherwise caused turbidity in the solution. A similar experiment was carried out in 8 M urea. The number of tryptophyl residues oxidized was calculated from the empirical relationship (11) 1 Obtained from MO och Domsjij AB, Ornskiildsvik, Sweden. Batch 4113. Molecular degree of substitution (MS) = 0.51.
777
OF A CELLULASE
n=
1.31’A&& 5500 X molarity
cellulase
’
where AAzso is the maximum difference in absorbance. Reaction of the tryptophyl residues with 2-hydroxy-5-nitrobenzylbromide (Z Br) was performed as described by Horton and Koshland (12). To 2 ml samples of cellulase (A280 = 2) in 0.1 M sodium acetate buffer pH 5.0, aliquots of # Br dissolved in dried aceton were added to a final molar ratio of 2O:l (6 Br:tryptophan). A small aliquot of the sample was withdrawn and tested for enzymic activity. The reaction mixture which contained some precipitate was then dialyzed for 2 hr against 0.1 M glycine-NaOH buffer pH 10.5, 8 M urea. The solution, which now was free from precipitate was run on a Sephadex G25 column 1.5 X 20 cm in the same buffer. The protein fractions were collected and the number of 2-hydroxy-5-nitrobenzyl groups covalently bound was estimated from the absorbance at 410 nm assuming a molar extinction coefficient of 18,000 (12). Nitration of tyrosyl residues was carried out using tetranitromethane TNM as described by Sokolovski et al. (13). A!iquots of a 0.84 M solution of TNM in 95y0 ethanol were added to l-ml samples of protein. One series of samples contained native enzyme while the other contained heat denatured enzyme obtained by incubation at 80” for 15 hr. The reaction mixture was left for 4 hr at room temperature (22’) and was then gel filtered on a Sephadex G-25 column (1 X 15 cm), in 0.05 M Tris-HCl buffer pH 8.0. The protein fractions were pooled and tested for enzyme activity. The extent of modification was estimated by measuring the absorbance at 428 nm assuming a molar extinction coefficient of 4100 (13) for 3-nitrotyrosine. In another experiment, samples of native and heat denatured cellulase were modified according to the procedure described using a molar excess of reagent (TNM: tyrosyl residues) of 80. After modification the protein was hydrolyzed for 24 hr with 6 M HCl at 110” and subjected to amino acid analysis. Coupling with diazonium-1-H-tetrazole (DHT) was carried out as described by Horinishi et al. (14) and as modified by Sokolovsky and Vallee (15). DHT was prepared from amino-1-H-tetrazole (14). The concentration was determined by measuring the formation of the monoazoderivative of N-acetyltyrosine (15). The coupling of DHT with the enzyme was performed in 0.67 M NaHC03 pH 8.8 at room temperature. The extent of reaction was followed by measuring the absorbances at 480 nm (biazohistidine) and 550 nm Activity measurements were (biazotyrosine). carried out along with the coupling reactions.
778
PETTERSSON
Succinylation was performed by adding solid succinic anhydride stepwise to a solution of cellulase in 0.2 M potassium phosphate buffer pH 7.5. The protein concentration was usually 1 mg per ml. After each addition of succinic anhydride the pH was checked and if necessary adjusted to the original value with 0.1 M NaOH. The final molar excess (succinic anhydride:amino groups) was 100. The extent of succinylation was determined with the ninhydrin method according to Moore and Stein (16) using phenylalanine as a standard. To study the apparent accessibility of tryptophan groups the neutral solvent perturbation technique by Herskovits and Laskowski (17) was applied. Sucrose and cellobiose were used as perturbants. Two well-matched l-cm tandem silica cells were used. The protein concentration was approximately 0.5 mg per ml. Spectra were recorded at the following pH values, 2.1, 2.9, 4.1, 5.0, 6.0, 7.1, and 8.1. Using the following buffer systems: HCl-KCl, glycine-HCl, NaAc-HAc, Tris-HCl and glycine-NaOH. The ionic strength was 0.05. The dependence of the intensity of tryptophan fluorescence upon the level of perturbant (18) was determined using propylene glycol as neutral perturbant. Native cellulase was compared with a peptic digest. The peptic digestion was carried out as follows: To 1 ml of cellulase dissolved in 0.01 M HCl, an aliquot of pepsin in 0.01 M HCl was added so that the molar ratio of pepsin:cellulase became 1:lOO. The mixture was left for 15 hr at 37”. The reaction was interrupted by adjusting the pH to 8 with sodium hydroxide. The wavelength of excitation was 290 nm and the wavelength of maximal emission was near 350 nm. Materials. The cellulase was purified as described in (1). 2-Hydroxy-5-nitrobeneyl bromide was obtained from Nutritional Biochemical Corporation, Cleveland, Ohio, USA. Teranitromethane was obtained from Fluka AG, Switeerland and N-acetyl tryptophanamide from Mann Research Laboratories, New York, N.Y., USA.
0x1~~4~10~ OF TRYPTOPHYL WITH NBS
RESIDUES
Oxidation of tryptophyl residues with KBS has been shown to be a specific and convenient method for determining the tryptophan content in proteins (11). The method is based upon t#hedecrease in absorbance at 280 nm accompanying the transformation of the indole to the oxindole chromophere. ii titration of the tryptophyl residues of native cellulase at pH 5 is shown in Fig. 2 (open circles). The initial linear decrease in absorbance with increasing concentration of NBS is followed by a region with nearly constant absorbance. This region actually fol-
2
4
6
6
PH FIG. 1. Effect of pH on the activity of a cellulase from Penicilliunz not&urn. Viscometric deSubstrate: 0.4yo hydroxyethylceltermination. lulose. Enzyme concentration Azgo = 1 X 10-a. Temperature 25”.
RESULTS
DETERMINATION OF THE PH-OPTIMUM OF THE ENZYME
Due to its higher precision the viscometric assay method was chosen in this case. To avoid complications due to protolysis of the substrate hydroxyethylcellulose was used instead of carboxymethylcellulose. The relative cellulase activity B (10) as a function of pH is given in Fig. 1. The curve has a broad maximum in the region pH 4.57 and falls rapidly on both sides of this optimum.
FIG. 2. Oxidation of the tryptophans of cellulase with 0.001 M NBS. Open circles: represent the ratio of decrease in absorbance at 280 nm to the initial absorbance. Solid circles : percent inactivation. Concentration of cellulase: 0.25 mg/ml. Medium: 0.1 M HAc-NaAc pH 5.0. Room temperature (22”).
STRUCTURE
AND FUNCTION
779
OF A CELLULASE
denaturing medium (8 N urea) only 2.7 residues were modified. Activity measurements on the modified product revealed that 60% of the initial activity was recovered. ACCESSIBILITY
10 NBS/cellulase
20 mole/mole
FIG. 3. Oxidation of the tryptophans of cellulase with 0.01 M NBS in 8 M urea. Open circles: the ratio of decrease in absorbance at 280 nm to the initial absorbance. Concentration of cellulase: 0.25 mg/ml. Medium: 0.1 M HAc-NaAc, 8 M urea. Room temperature (22”).
lows by one with a slow increase in absorbance due to opalescence. The plateau value corresponds to the titration of 2.3 tryptophan groups. From Fig. 2 it may also be seen that the activity (filled circles) decreases concomitantly with the oxidation of tryptophyl residues, and that the plateau value for complete inactivation roughly coincides with the plateau value for the titration curve. If the titration with NBS is carried out in a denaturing medium (8 M urea) almost all the tryptophyl residues are titrated (Fig. 3). The total decrease in absorbance corresponds to the oxida,tion of 12 tryptophyl residues. The total tryptophan content of the cellulase has previously been determined to be 13 residues per mole. SUBSTITUTION WITH
OF TRYPTOPHYL
OF TRYPTOPHYL
RESIDUES
The neutral solvent perturbation technique of Herskovits and Laskowski (17) was used to examine the perturbation effects of cellobiose, which is a product of the enzyme action and probably an inhibitor, and sucrose which has no inhibitory effect. Perturbation spectra were recorded at pH values from 2.1 to 8.1. A suboptimal concentration of perturbant (10%) was used due to the limited solubility of cellobiose. The perturbation spectrum of cellulase with 10 % cellobiose at pH 4.1 is shown in Fig. 4 (open circles). The main optimum at 292 nm arises entirely from the perturbation of the typtophan spectrum while tyrosyl groups may ha,ve an influence on the peak at 285 nm. The perturbation effect obtained with 10% sucrose under the same conditions is much lower (Fig. 4, filled circles) but the optima appear at approximately the same wavelengths. A control experiment with N-acetyl-n-tryptophanamide showed no significant difference in perturbation effect between cellobiose and sucrose. The perturbation spectra had the same general features over the pH interval studied. The pH dependence of AEM at 292 nm is shown in Fig. 5 with cellobiose (open circles) and sucrose (filled circles) as perturbants. The cellobiose curve has a maximum in the region pH 4-6 while the sucrose curve has its minimum in the pH region 3-5.
RESIDUES
2-HYDROXY-5-NITROBENZYLBROMIDE
2-Hydroxy-5-nitrobenzylbromide p Br is a reagent with a high degree of specificity towards tryptophyl residues in acid and neutral media (12). The only other amino acid that reacts is cysteine (12). With a final molar ratio of 2O:l (9 Br: tryptophan) the extent of modification was determined to be 4.6 residues per mole cellulase. The extent of modification was constant even if the molar excess of reagent was increased to 50: 1. When the reaction was carried out in a
270
280
290
304
A nm
FIG. 4. Perturbation spectra of cellulase. Media: 10% cellobiose, open circles and 10% sucrose, solid circles. Protein concentration: 0.5 mg/ml, pH 4.1.
780
PETTERSSON
2.0 0
0
‘: q.2 w lu
a
0.4
D
PH FIG. 5. The pH profile of the height of the perturbation peak at 292 nm (Aen&. Media: 10% cellobiose open circles, and 10% sucrose solid circles. Protein concentration: 0.5 mg/ml.
D f 8 : :: t 20 0 3 E : .z 30 ‘$ 10 or”
E Percent
10 20 propylene glycol
30
FIG. 6. The intensity of tryptophan fluorescence at 350 nm as a function of concentration of propylene glycol. Open circles: native cellulase, solid circles: a peptic digest of the cellulase. Excitation wavelength 290 nm. pH 5.0. Protein concentration: 0.03 mg/ml.
Fluorescence studies. The accessibility of tryptophyl residues to neutral solvent perturbants has been studied by Steiner et al. (18) by measuring the dependence of fluorescence upon the concentration of perturbant. This method was adopted to examine the accessibility of the tryptophyl residues of the cellulase. As neutral solvent perturbant propylene glycol was used and as standard a peptic digest. In Fig. 6 the relative Auorescence intensity is plotted as a function of the concentration of propylene glycol. The intensity of fluorescence of the cellulase increases linearly with the concentration of propylene glycol and the slope is 29 % of that of the peptic digest. The figure 29% means that 3.8 tryptophans out of 13 behave as if they were exposed to the perturbant. This figure may however have a very limited
quantitative significance owing to the fact that different tryptophyl residues can have different quantum yields. The method described above was also used to check the perturbation effect of cellobiose and sucrose. Though these experiments have not yet been terminated it is evident that there is no significant difference between cellobiose and sucrose as perturbants in this case. Nitration of tyrosyl groups. Tetranitromethane (TNM) has been shown to be a mild and specific reagent for the nitration of tyrosyl groups in proteins (13). If increasing amounts of TNM are added to native cellulase the nitration of tyrosyl groups increases until a plateau is reached (see Fig. 7, open circles). The plateau value corresponds to the nitration of 4 to 5 tyrosyl groups. The enzymic activity decreases concomitant with the nitration of tyrosyl groups (Fig. 7, squares) and reaches a plateau region at about 35 % of the initial activity. At higher concentrations of TNM there seems to be a decrease in activity again. In heat-denatured cellulase, 11-12 tyrosyl residues are nitrated. This plateau level is reached very rapidly as can be seen from Fig. 7 (filled circles). Samples of native and heat-denatured cellulase were nitrated with TNM using a molar excess of 60: 1. The samples were hydrolyzed and subjected to amino acid analysis. A
0
J 20 40 60 60 100 TNM/ tyrosyl residues mole/mole
FIG. 7. Nitration of cellulase with TNM. The formation of 3-nitrotyrosyl residue and the effect on the enzymic activity of increasing amounts of TNM. Open circles: native enzyme. Solid circles: heat denatured enzyme. Squares: Cellulase activity. The reaction was performed at pH 8.0 at room temperature. The degree of nitration was determined spectrophotometrically at 428 nm.
STRUCTURE
AND
FUNCTION
781
OF A CELLULASE
new peak appeared in the chromatogram at the same place as 3nitrotyrosine (13). In the case of denatured enzyme the tyrosine peak almost completely disappeared which indicated that all tyrosyl residues were actually modified. In the case of native cellulase the tyrosine peak was reduced corresponding to the nitration of 5 tyrosyl residues. COUPLING WITH DIAZONIUM-1 II-TETRAZOLE DHT
20 OHT lcellulose
The coupling reagent diazonium-l-Htetrazole DHT was introduced by Horinishi and co-workers (14) for spectrophotometric determination of histidine in proteins. The method has later been modified by Sokolovsky and Vallee (15). Contrary to the other reagents used in this work DHT is very unspecific and apart from histidine and tyrosine it also reacts with amino and hydroxyl groups. Figure 8 shows the absorbances at 480 (biozohistidine, open circles) and 550 nm (biozotyrosine, filled circles) as a function of -the concentration of DHT. The absorbance at 480 nm increases rapidly at first then more slowly. -4 true plateau value is never reached in spite of the very high excess of reagent used. It may also be seen from Fig. 8 that the absorbance at 550 nm also increases almost linearly upon addition of DHT. It is thus evident that biazotyr0sin.e or possibly monoazotyrosine, which also has some absorbtion at 550 nm (15) f orms concomitant with biazohistidine and that it is not possible to determine histi-
0.5
5 5 0.3 G 6 f :: 0.1 9
0.5
E 0.3 % Y) % P 0 s
0.1 2 a
40
60
mole/mole
FIG. 9. The effect of increasing amounts of DHT on the cellulase activity. Coupling condi-
tions: bicarbonate buffer pH 8.8, reaction time 30 min, room temperature, protein concentration 0.2 mg/ml.
dine independently of tyrosine before all histidyl and tryosyl groups have reacted to form the biazoderivatives. The enzyme is rapidly inactivated upon addition of DHT (Fig. 9) and the inactivation is practically complete at a molar excess of 60, (DHT: enzyme) \vhich is before any significant amounts of colored products have been formed. SUCCINYLATION WITH SUCCINIC ANHYDRIDE
Succinic anhydride reacts specificly with amino groups but under specific conditions also with hydroxyl groups (19). If native cellulase is treated with succinic anhydride with a molar excess of approximately 100 (succinic anhydride: amino group) about 60 % of the amino groups are modified. This figure was obtained after determination of the amino groups with ninhydrin and is of course only a rough estimate. It is to be observed that the degree of substitution was roughly independent of the molar excess of reagent in the region 20-200 (succinic anhydride: amino group). The enzymic activity was uneffected or increased somewhat upon succinylation. INHIBITION STUDIES WITH RIBOFLAVIN5'-PHOSPHATE AND HISTAMINE
FIG. 8. Coupling of DHT with cellulase as determined by the absorbance at 480 and 550 nm. Coupling conditions: bicarbonate buffer pH 8.8, reaction time 30 min, room temperature, protein concentration 0.2 mg/ml.
Riboflavin-5’-phosphate has been shown to form red complexes with free indole compounds and with tryptophan bound in proteins (20). Complexes of the charge trans-
782
PETTERSSON
dized with NBS before precipitation occurred, and that 4.6 could be modified with p Br. The fact that only 2.7 residues were modified with $?JBr in the urea-denatured see text.) enzyme seems nevertheless to indicate that some other factsor than difference in exposure activity Cont. of inhibitor (M) Enzyme Inhibition (% of initial) of the tryptophyl residues was limiting in this case. It is to be observed that 12 tryp81 1.0 x 10-z RFP 72 2.0 x 10-z tophyl residues were titrated with NBS if the reaction was carried out in 8 M urea. 74 Histamine 1.0 x 10-t Further evidence for the limited accessibility 68 2.0 x 10-z of the tryptophyl groups have been achieved 51 8.0 X 1O-2 from the spectrofluorometric measurements (see Fig. 6). Oxidation of 2.3 tryptophans fer type have also been identified between with NBS caused complete inactivation of imidazole and indole compounds. Moreover the enzyme. From Fig. 2 it may be seen that imidazole derivatives have been found to be there is a good parallelism between the activinhibitors of lysozyme (21). The results given ity curve and the decrease in absorbance at in Table I show that riboflavin and histamine 280 nm. This may indicate that no other are weak inhibitors of the cellulase activity. amino acid side groups have reacted beside the tryptophyl residues. The low consumpDISCUSSION tion of NBS, 7 moles per mole of proThe pH profile of an enzyme reaction may tein points in the same direction. The be used for identifying the groups on the modification of 4.6 tryptophyl residues with enzyme molecule that are involved in the @ Br caused only a 40 % inactivation. If one catalytic mechanism. Generally a determinasupposes that the same tryptophans have tion of the pH dependence of the Michaelis reacted, which of course is not certain, these parameters V and K, as well as the pH results seem to contradict each other. On the stability is needed to properly exploit the other hand it is reasonable that oxidation possibilities of this method (22). The pH of indole groups to oxindole derivatives may profile of the cellulase may be seen in Fig. 1. cause a greater change in the sturcture This is a composite curve which partly may around the indole group than int,roduction depend on changes in K, and partly on of a benzene ring. It is as a matter of fact changes in V. Though initial velocities have astonishing how sensitive many enzymes are been measured it is not ruled out that the to oxidation with NBS (11) even at a rather fall in activity on the acidic side of the optilow excess of reagent where the risk for s&tmum may be due to an irreversible con- ting peptide bonds is minimal. formation change. It has been previously Cellobiose, which is a product of the found that the stability of the enzyme de- enzyme reaction was tried as a neutral percreases rapidly at pH values below 3 (3). The turbant using the difference spectrophotomfall in activity on the alkaline side might etry method according to Herskowits and depend on a change in the state of ionization Laskowiski (17). It was found that cellobiose of a group with a pK, value of 7.5 (see Fig. gave rise to a typical tryptophan spectrum 1). This might be an amino or more probably and that the perturbation effect was much an imidazole group. The substrate is com- more pronounced than with sucrose as perpletely non-ionic and should thus have no turbant (see Fig. 4). Moreover the pH profile effect on the pH profile. of the difference peak at 292 nm (Fig. 5) The enzyme contains 13 tryptophyl resi- displays a close resemblance with the pH dues of these only a few seem to be exposed. activity curve (Fig. 1). The corresponding This has been found earlier from difference curve for sucrose has quite a different spectrophotometric measurements and has appearance (Fig. 5). It must be emphasized now been further elucidated. It was found that that these results do not necessarily mean approximately 2 tryptophans could be oxi- that there is a specific interaction between TABLE
INHIBITION
I
OF A CELLULASE FROM notatum WITH RIBOFLAVIN-5’-PHOSPHATE AND HISTAMINE (For experimental
Penicillium RFP details
STRUCTURE
AND FUNCTION
cellobiose and tryptophyl residues on the protein. It is also possible that cellobiose can perturb trypt’ophyl residues not accessible to other perturbants by some specific mechanism, without the presence of any direct binding. If the active site of the enzyme is in a cleft as in lysozyme (7) it is possible that cellobiose has the ability to penetrate into this cleft either directly or by inducing a conformation change. Another indication of a possible role of the tryptophyl residues in the enzymatic mechanism can be shown from the inhibition experiments with riboflavin and histamine. These substances have been shown to form complexes of the charge transfer type with tryptophyl residues in proteins (20, 21). Moreover histamine is an inhibitor of l-ysozyme (21). It is seen from Table I that iiboflavin and histamine inhibit the cellulase, though a rather high concentration is required. From spect’rophotometric titrations it was concluded that the tyrosyl groups titrated freely and with the same pK (3). Since conformation change occurs before the titration of the tyrosyl groups (3), this does not rule out the possibility that some tyrosyl groups might be unexposed in the native molecule. The fact that only 5 tyrosines out of 15 were nitrated with TNM seems to support this hypothesis (Fig. 7). On the contrary after heat denaturation 11-12 tyrosines were nitrated as determined with direct spectrophotometry (14). Amino acid analysis on the other hand revealed that all tyrosyl groups were nitrated. The difference between the two methods was probably due to the limited precision in the spectrophotometric method. Nitration of 5 tyrosyl groups was associated with a loss of 65 % of the initial act,ivity. Considering the fact that nitration causes a shift to lower pH values of the pK of tyrosine it is possible that this loss in activity is due to a change in the stability of the enzyme. Coupling with DHT (diazonium-IH-tetrazole) caused :a complete loss of activity (Fig. 9) at a very low excess of reagent. This happened before any biazoderivatives had been formed as judged by measurements of the absorbance at 450 and 550 nm (15). Since DHT is a rather unspecific reagent it is
OF A CELLULASE
783
difficult to draw any conclusions about the reason for the inhibition. Nevertheless histidy1 groups are probably more reactive than other possible groups. The inhibition might thus be due to formation of monazohistidyl derivatives. 60 % of the amino groups could be succinylated with succinic anhydride. This figure was determined from the reduction in ninhydrin color and must be regarded as a rough estimate. Succinylation did not effect the enzymatic activity. Data suitable for a comparison with the present findings are still, with a few exceptions, rather scanty in the literature on cellulolytic enzymes. Generally fungal cellulases seem to have pH optima on the acid side of neutrality (23). Li et al. (24) found a broad pH optimum similar to that described here for a highly purified endoglucanase from Trichoderma viride. Similar pH optima have also been observed for two highly purified cellulases from Stereum sanguinolentum studied by Bjiirndal and Eriksson (25). Inhibition with NBS has been found by Iwasaki et al. (26) studying two cellulases from Trichoderma koningi. The further work on this enzyme will concentrate on the role of tryptophyl and histidy1 groups for the enzyme action. Moreover the kinetics and mode of action on different cellulose substrates will be studied. ACKNOWLEDGMENT I thank my teacher Professor J. Porath for his kind interest throughout the progress of this work and Mrs. Ulla-Britt Fredriksson for skilful technical assistance. The work was supported by a grant from the Hierta-Retzius foundation. REFERENCES 1. PETTERSSON, G., Arch. Biochem. Biophye. 133, 307 (1968). 2. PETTERSSON,G. AND EAKER, D.L., Arch.Biothem. Biophys. 124, 154 (1968). 3. PETTERSSON, G. AND ANDERSSON, L., Arch. Biochem. Biophys. 134,497 (1968). 4. ERIKSSON, K.-E. AND PETTERSSON, G., Arch. Biochem. Biophys. 134, 160 (1968). 5. NORKRANS, B., Adv. Appl. Microbial. 9, 91 (1967). 6. PERUTZ, M. G., Prac. Roy. Sot. (London) Ser B. 16’7, 348 (1967).
7. BL.~KE, C. C. F., JOHNSON, LOUISE N., MUIR, G. A., NORTH, A. C. T., PHILIPS, D. C. AND SARMA V. R., Proc. Roy. Sot. (London) Ser. B. 167, 378 (1967). 8. RUPLEY, J. A., Proc. Roy. Sac. (London) Ser. B. 167, 416 (1967). 9. PETTERSSON, G. .~ND PORATH, J., in “Methods in Enzymology,” VIII, p. 603, New YorkLondon 1966. 10. ALMIN, K. E. END ERIKSSON, K.-E., Biochim. Biophys. Acta 139, 238 (1967). 11. SPANDE, T. F. -YND WITKOP, B., in “Methods in Enzymology,” XI, p. 498, New YorkLondon 1967. 12. HORTON, H. R. AND KOSHLAND, I). E., JR., J. Am. Chem. Sac. 87,1126 (1965). 13. SOKOLOVSKY, M., RIORD~N, J. F. AND VALLICE, B. L., Biochemistry 6, 3582 (1966). 14. HORINISHI, H., HXHIMORI, Y., KURIHaRa, K. AND SHIB.ITA, K., Biochim. Biophys. Acta 86, 477 (1964). 15. SOKOLOVSKY, M. AND VALLEE, B. L., Biochemistry 6, 3574 (1966). 16. MOORE, S. AND STEIN, W. H., J. Biol. Chem. 176, 367 (1948).
17. HEKSKOVITS, T. T. .\ND ~~~~~~~~~~~~ M. JR., J. Biol. Chem. 237, 2481 (1962). 18. STEINER, 11. F., LIPPOLDT, R., EDI~:LHOCH, H. BND FRBTT~LI, V., Biopolymers Symp. 1 p. 355 (1964). 19. TICRMINIELLO, L., BIER, M. AND NORD, F. F., Arch. Biochem. Biophys. 73, 171 (1958). 20. ISENBERG, I. BND SZENT-GY~RGYI, A., Proc. Alat. Acad. Sci. U.S.A. 44, 857 (1958). 21. SHINITZKY, M., KAT~H~LSKI, E., GRISARO, V. AND SH.IRON, N., Arch. Biochem. Biophys. 116, 332 (1966). 22. DIXON, M. racy WEBB, E. C. “Enzymes,” p. 116. Longmans, Green and Co. Ltd. 2nd Ed. London 1964. 23. G.~SCOIGNX, J. A. AND GBSCOIGNE, M. M., “Biological Degradation of Cellulose,” p. 58. Butterworths, London 1960. 24. LI, L. H., FLORI, R. M. AND KING, K. W., Arch. Biochem. Biophys. 111,439 (1965). 25. BJ~RNDAL, H. AND ERIKSSON, K.-E., Arch. Biochem. Biophys. 124, 149 (1968). 26. 1w.4~~1, T., IKEDA, R., H~Y~SHI, K. AND FUN.~TSU, M., J. Biochem. (Japan) 67, 478 (1965).