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Kinetics and mode of action of Aspergillus niger polygalacturonases
J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.
Kinetics and mode of action of
221
AspergiUus niger polygalacturonases
Jacques A.E. Benen, Harry C.M. Kester, Lucie Parenicovfi and Jaap Visser. Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreyenlaan 2, 6703 HA Wageningen, The Netherlands.
Abstract Endo-polygalacturonases I and II (PGI and PGII) isolated from recombinant A. niger were characterized with respect to pH optimum, activity on polygalacturonic acid (pga), mode of action and kinetics on oligogalacturonates. Vmax and Km values using pga as a substrate at the optimum pH 4.1 were calculated as 500 U/mg and 0.15 mg/ml and 2000 U/mg and 0.15 mg/ml for PGI and PGII, respectively. Mode of action analysis revealed a random cleavage pattern for PGII while for PGI multiple attack on a single chain was observed. For PGII a partial subsite map was obtained. Site directed mutagenesis of His223 of PGII with subsequent analysis of the mutated PGII revealed that His223 is essential for catalysis.
Introduction Polygalacturonases from primarily fungal origin have been studied since several decades. Nowadays many genes encoding polygalacturonases, both exo- and endo-acting, from numerous different species have been cloned. Despite the large number of genes available and the long record of polygalacturonase studies most studies were directed at the purification of the enzymes and a limited characterization comprising mostly whether the enzyme is exo- or endo-acting, the activity on polygalacturonic acid and the pH, temperature and ionic strenght optima while very few studies were carried out toward the understanding of the mode of action, determined by the characteristics of the individual subsites of the enzymes. First studies addressing this problem via the determination of the number of subsites of an Aspergillus niger polygalacturonase and identification of catalytically important residues of this enzyme were described by Rexov~i-Benkovfi [1, 2]. Unfortunately, these studies have found only little follow up by other research groups [3]. Therefore detailed knowledge about 'subsite architecture' of these industrially important enzymes is scarce. Bussink et al. [4] and Kusters-van Someren et al. [5] have shown that in A. niger for both polygalacturonases (PGs) and pectin lyases (PLs) families of genes are present: seven
222 PG and six PL encoding genes were identified [4, 5]. The occurrence of families of genes encoding different enzymes raises the possibility of a concerted action of the enzymes of one or both families in the degradation of pectin. In order to clarify the role of the individual enzymes in the pectin degradation a comparative study in this respect was initiated. For this, individual genes were fused with the pkiA promoter of the glycolytic pyruvate kinase gene that allows expression of the individual genes under conditions where all other pectinases are repressed. Careful analysis of mode of action, kinetic parameters and subsite affinities of the enzymes on model and natural substrates will reveal the role of the individual enzymes in pectin degradation. Here we report on the characterization of recombinant PGI and PGII, the two most abundant PGs in the commercial Rapidase preparation [3] and of a site specific mutated PGII in which His223 was changed into Ala.
Materials and Methods Molecular biology. All DNA manipulations were performed using standard techniques. Promoter-gene fusions were constructed as described by Kusters-van Someren et al. [6]. Site directed mutagenesis of His223 of PGII was carried out in essentially the same way. PCR generated DNA fragments were checked for undesired mutations by sequence analysis. Transformation of A. niger NW228 (pyr, prtF) with appropriate plasmids was done as described before [7]. Growth and purification. A. niger strains transformed with either the pki-pgaI or pla'-pgalI fusion were grown in batch in 1L flasks containing 350 ml minimal medium according to Pontecorvo et al. [8] supplemented with Vishniacs spore element solution, 0 . 1 % yeast extract, 70 mM NH4C1 and 4 % fructose as a carbon source. Cultures were inoculated with 1 • 106 spores/ml and grown at 30 ~ in a rotary shaker for 18 hrs. Mycelium was separated from the medium by filtration over a nylon membrane. The filtrate was adjusted to pH 6.0 and loaded onto a DEAE-Sepharose Fast Flow column pre-equilibrated at pH 6.0 (10 mM Bis-Tris/HC1). Elution was performed with a linear gradient, 0-600 mM NaC1, in 10 mM Bis-Tris pH 6.0. SDS-PAGE of individual fractions demonstrated that the enzymes were pure after this separation. Fractions containing the enzyme were pooled and dialysed against 50 mM Na-acetate pH 4.5 and stored at either -20 ~ or 4 ~ until use. Mode of action and kinetics. Routine polygalacturonase assays were performed in a reaction mixture containing 50 mM Na-acetate pH 4.2 and 0.25 % w/v pga at 30 ~ The release of reducing sugars was determined according to Stephens et al. [9]. For determination of pH optima the 50 mM Na-acetate buffer was replaced by Mcllvain buffers. For the determination of kinetic parameters and for the mode of action of the enzymes reaction products were analysed on a Dionex BioLC/high-performance chromatography system using a Carbo Pac PA-100 anion-exchange column (25 cm x 4 mm) with pulsed amperometric detection. The samples loaded were eluted with a linear gradient of 0.15-0.60 M Na-acetate in 0.1 M NaOH at 1 ml/min in 22 min. Products were quantitated via calibration mixes containing oligogalacturonates with DP 1-8 (G1-G8) at 0.1 mM each and via an internal standard of 0.1 mM glucuronic acid (eluting between G1 and G2) with 50 pl injections.
223
Results and Discussion The PGI and PGII produced from strains transformed with the promoter gene fusion are in all respects tested identical to those enzymes obtained from the wild type strain when grown on pectic substances. For both PGI and PGII the pH optimum is 4.1-4.2 in 50 mM Na-acetate buffer, 30 ~ All further kinetic analyses were performed under these conditions. PGII. Using pga as a substrate Km and Vmax of PGII were calculated as 0.15 mg/ml and 2050 U/mg, respectively. The high Vmax and low Km demonstrate that pga is a good substrate for PGII. By following the product formation as a function of time (Fig. 1) it was demonstrated that PGII is a randomly cleaving endo polygalacturonase. The progression of substrates is characterised by an initial transient increase of higher oligogalacturonates which are gradually converted to oligomers with lower DP, eventually resulting in a mixture of G1, G2 and G3. The rather strong transient increase of G4 and G5 is not a result of transglygosylation, since PGII is an inverting enzyme (see Biely et al. elsewhere in this volume), but is merely due to the slow hydrolysis of these compounds as will be discussed below.
G2
0.4
G3
G1 IS
G4
0.3 90 min
~- 0.2 _
-
-
-
35 min
0.1
J A
0.0 0
5
~ _ . _ . _ . ~ _ A
10
15
10 min I 20
minutes
Figure 1. HPLC analysis of product progression during hydrolysis of 0.25 % polygalacturonate by PGII. Aliquots were withdrawn from the reaction mixture at timed intervals and reactions were stopped by raising the pH of the sample to pH 8.0 by mixing with 1 volume 25 mM Na-phosphate pH 9.5. G1 to G5 indicate the oligogalacturonates with corresponding degree of polymerization. The vertical axis shows the responce of the pulsed amperometric detector and the horizontal axis the elution time. Times of sampling are indicated above the trace.
224
Table I. Mode of action of PGII. Bond cleavage frequencies (BCF) in percentage for oligogalacturonates. The reducing or end of the products is indicated with a solid circle. The position of cleavage is indicated with a solid triangle. DP
BCF (%)
4 5
6
o
o
o o
Products
o
o
o
9
100
o o
o o
o o
9 o
9
67 33
dimer
o o o
o o o
o o o
9 o o
9 o
35 57 8
dimer trimer
9
A
In order to estimate the number of subsites, the binding affinities, the location of the active site and the cleavage patterns reduced and non reduced oligogalacturonates of DP 4 to 6 were used as substrates and the resulting products analysed by HPLC and TLC. Table I lists the bond cleavage frequencies for PGII. G4 is exclusively split in 1-3 mode while reduced G4 was not hydrolysed. G5 is cleaved in the 1-4 and 2-3 mode at 67 % and 33 % respectively, while reduced G5 is only split into reduced G2 and G3. The reduced G6 is not cleaved in the 1-5 mode, while reduced G2 and reduced G3 are readily formed. The non-reduced G6 is cleaved in 1-5, 2-4 and 3-3 modes yielding equimolar product pairs as was also seen for the cleavage of G4 and G5. These data demonstrate that cleavage of the glycosidic bond occurs from the reducing end. In time course experiments using different oligogalacturonates at several concentrations the turnover numbers and Km values were estimated for each oligomer in the individual binding modes and used for the calculation of the thermodynamic parameters of PGII according to Thoma et al. [10] and Hiromi et al. [11]. In Table II the data are presented. An approximation of the intrinsic rate constant, kint, was calculated from Vmax on pga (kint = kcat). From kint and Km and the turnover number for each binding mode the binding energies for each mode were calculated which were in turn used for the calculation of the individual subsite affinities listed in Table III. Since G4 is the smallest substrate used in this study, it is not possible to obtain information of the subsites at positions -3 to 1.
225 Table II, Kinetic and thermodynamic parameters of PGII using oligogalacturonates as substrates. Kp was calculated from Kp=[ko/Km]/kint while kint was obtained as described in the text. AG was calculated from -AGp=RTlnKp + 10 kJ/mole. Mode indicates the cleavage mode DP
Mode
Km • 10-6 (M)
ko
ko/Km x 10-6 Kp
(s -1)
(M -1 s-1)
(M -1)
AGp (kJ/mole)
4
G3 + G1 22
103
4.7
8048
-22.6
5
G4 + G1 13 G3 + G2 25
315 159
24.2 6.4
41438 10960
-26.7 -23.4
G5 + G1 40 G4 + G2 16 G3 + G3 31
148 386 56.5
3.7 24.1 1.8
6336 41267 3082
-22.0 -26.7 -20.2
Table III. Subsite affinities Ai for PGII (i denotes the subsite number). Subsites with a '-' prefix are located towards the non-reducing end of the substrate while subsites with a ' + ' prefix are located towards the reducing end. The active site is located between subsites -1 and + 1. Subsites -3 to + 1 were determined as one value. Subsite (Ai) Affinity (kJ/mole)
-4 +4.2
-3 / + 1 +22.6
+2 +0.8
The subsite map and the data on reduced oligomers, which showed formation of reduced G2 on reduced G5, indicate that the number of subsites is 5, stretching from position-4 to 1. The sum of the individual turnover numbers for each oligomer also shows that the rate of hydrolysis of the oligomers with DP4 and DP5 is much slower than of those with higher DP which is reflected in the product progression curves in Fig. 1. Rexov~i-Benkov~i [1] studied an A. niger endopolygalacturonase which might have been the same as the PGII described here; the cleavage pattern described is very much the same as found here and the pH optimum is exactly the same. The number of subsites for that enzyme was found to be four which is not in agreement with the number found for PGII. However, bearing in mind that in the study of Rexovgt-Benkovgt the individual binding modes of each oligogalacturonate were not addressed and therefore no subsite map was obtained the additional fifth subsite might have been overlooked.
226 PGI.
In a similar way as for PGII the kinetics for PGI were addressed. Using pga as a substrate Km and Vmax were 0.15 mg/ml and 500 U/mg, respectively. Fig. 2 shows the product progression upon pga hydrolysis. There is a strong increase of G1, G2 and G3 which is accompanied by a transient increase of G4 and G5 while there is only a small increase of oligomers with DP higher than 5. The transient increase of G4 and G5 is not due to transglycosylation since like PGII, PGI is also an inverting enzyme (see Biely et al. elsewhere in this volume). The profiles of PGI suggest that the enzyme after first random cleavage of the polymer substrate degrades the higher oligomers formed preferentially via hydrolysis of terminal residues at the reducing end. The latter was demonstrated by hydrolysis of reduced oligogalacturonates while the former was investigated in more detail by analysing the mode of action and cleavage rates on defined oligomers ranging from DP 4 to 8 . PGI hydrolyses G4 mainly to G1 and G3 at equimolar amounts as expected and a small amount is digested into G2 (Results not shown). G5 is cleaved to G1 and G4 in again equimolar amounts and to almost the same extent to G2 and G3 also in equimolar amounts (Results not shown). Thus, for G4 and G5, PGI is not much different from PGII, only the bond cleavage frequencies being slightly different. Upon hydrolysis of G6 to G8 the formation of equimolar product pairs is not observed anymore. With G6 as substrate G1 is formed at least twice as fast as G5 while G4 is formed
IS G2
0.14 0.12 0.10 0
0.08
G3 G4 G5
0.06 0.04
J
0.02
IS
0.00 0
G2 G3 G4 G5 I
I
10
15
10 min I 20
minutes
Figure 2. HPLC analysis of product progression during hydrolysis of 0.25 % polygalacturonate by PGI. Aliquots were withdrawn from the reaction mixture at timed intervals and reactions were stopped by raising the pH of the sample to pH 8.0 by mixing with 1 volume 25 mM Na-phosphate pH 9.5. G1 to G5 indicate the oligogalacturonates with corresponding degree of polymerization. The vertical axis shows the responce of the pulsed amperometric detector and the horizontal axis the elution time. Times of sampling are indicated above the trace.
227 faster as G2 (Fig 3). A similar behavior was observed by Robyt and French [12, 13] for cxamylase. They demonstrated that this type of product ratios, that differ from the beginning of the reaction, are due to multiple attack on a single chain. A ratio-plot according to Robyt and French [13] for G1 and G5 formation from G6 supported that PGI exibits multiple attack on G6 (not shown). The fact that still G5 is formed upon hydrolysis of G6 demonstrates that not all G6 bound in the G5-G1 mode is cleaved in a repetitive way. Thus, only a fraction of the substrate bound in this mode is subject to repetitive attack. The ratio between cleavage in this way and normal cleavage into G1 and G5 is determined by the dissociation constant of the G5 generated upon cleavage and the first order rate constant that is responsible for the shift of the bound G5 into the G4-G1 mode. Since still a considerable amount of G5 is formed, the dissociation constant of G5 and the first order 'shift' rate constant are of the same magnitude. However, from G7 and G8 as substrates it is clear that the ratio between the dissociation constant and 'shift' rate constant is completely in favor of shift when DP equals 6 or 7. With G7 as a substrate quite large amounts of G1 are formed while there is no detectable formation of the corresponding G6, only a rapid accumulation of G5 is observed. Similarly with G8 as a substrate quite large amounts of G1 and G2 are formed while there is no detectable formation of the corresponding G7 and G6, only again a rapid accumulation of G5 occurred which indicates that the multiple attack ceases when DP is down to 5 or 4, hence when dissociation is favored over shift. The previous data allow a clear interpretation of Fig. 2. PGI initially hydrolyses pga in a random endolytic way generating higher oligomers which is accompanied by rapid multiple attack of the higher oligomers in an exolytic way to yield mainly monomers and dimers from the reducing end. Therefore no transient accumulation of higher oligomers takes place. The appearance of transient G5 and G4 is due to the fact that the multiple attack of the higher oligomers stops when DP is 4 or 5 and these product are released. The transient accumulation of the tetramer partly originates from the particular binding mode of the higher oligomers. At present we are working on a kinetic model that describes the action pattern of PGI. It has been suggested that the multiple or single attack can be distinguished based on the relative rate of accumulation of mono- di- and trimers upon hydrolysis of polymeric substrate. At first glance this appears to be valid when comparing for example the mode of action of PGI and PGII (see Fig. 1 and Fig. 2). In a comparative study of three polygalacturonases by Pasculli et al [14] the enzymes were classified according these criteria while the rate of accumulation was by far not as clearcut differing as found for PGI and PGII. The observed profiles in that study can also be explained by assuming different rates of hydrolysis of the smaller (DP 4-6) oligogalacturonates. If for instance the rate of hydrolysis of G4 and G5 of PGII would have been higher then the progression curves would have more resembled those of PGI. The only way to discern between multiple or single attack is by analysis of the stochiometry of product pairs upon hydrolysis of oligomeric substrates at initial stages of reaction.
228 40 gl ,.i-,
o
(D c
30
.,.-~
(/) 0
20
g5
E 0c -
~c -
g4 g2 g3
10
~n-'~ 0
T 5
I I0
I 15
I 20
I 25
I 30
minutes
Figure 3. HPLC analysis of product progression during hydrolysis of 0.5 mM hexagalacturonate by PGI. Aliquots were withdrawn from the reaction mixture at timed intervals and reactions were stopped by raising the pH of the sample to pH 8.0 by mixing with 1 volume 25 mM Na-phosphate pH 9.5. G1 to G5 indicate the oligogalacturonates with corresponding degree of polymerization.
PGII His223Ala. Apart from mode of action and kinetics of wild type enzymes structure function relationships of these industrially important enzymes is of high interest to provide the necessary knowledge for genetic engineering of desired properties. As a first approach the identification of catalytically important residues was addressed in conjunction with the elucidation of the three dimensional structure [15]. Rexovfi-Benkov~i and Marckov~t [2] obtained evidence for the possible involvement of a histidine in catalysis of a fungal endopolygalacturonase. The same results were obtained for PGII using diethyl pyro-carbonate as a modifying agent in the presence or absence of substrate (Kester, unpublished). However chemical modification studies can never give conclusive evidence for the participation of the modified residue in catalysis or binding. The method to dissect the role of individual residues is site directed mutagenesis. Upon alignment of 25 endo- and exo-polygalacturonases taken from the swiss.prot. database only one histidine appeared to be conserved throughout. The conserved histidine at position 223 in PGII was changed into alanine, a small apolar residue. The mutation has a dramatic effect on Vmax using pga as substrate which decreases from 2050 U/mg to 10 U/mg; the Km did not change significantly, however. The mutation also affected the pH optimum of the enzyme as is shown in Fig. 4. The pH optimum narrows down to one pH unit. Since the polygalacturonate concentration used is well above the Km the apparent velocities can roughly be regarded as Vmax values at each pH. So, the fall and rise of the activity within one pH unit strongly suggests that catalysis is governed by only one ionisable group in the mutated enzyme, a glutamate or an aspartate. Thus, the
229
100 >,
80
60
~
9"~ rr
40 20 0 I
I
I
I
2.5
3
3.5
4
I
I
I
I
4.5
5
5.5
6
pH
Figure 4. Relative activities of wild type PGII and His223Ala mutated enzyme as a function of pH. Wild type enzyme, solid circles; His223Ala mutated enzyme, open circles. 0.30 0.25
' -
IIs
gl.
g3
0.20
G 0:=L
0.15 IS 0.10
g2
0.05
j~
0.00
I
0
5
i
I
,
10
!
!
15
20
minutes
Figure 5. Selected HPLC elution profile of products obtained after incubation of 0.25% polygalacturonate with PGII, upper trace, and PGII H223A, lower trace, respectively, demonstrating the effect of the mutation on catalysis. G1 to G3 indicate the peaks of the corresponding oligogalacturonates. IS indicates the internal standard, glucuronate. The vertical axis shows the pulsed amperometric detector response while the horizontal axis shows the retention time.
230 strong effect on Vmax, the absence of an effect on Km and the striking effect on the pH optimum, the shift in the direction expected, provide evidence for the involvement of His223 in catalysis rather than binding. This is again affirmed by the analysis of the products formed after hydrolysis of pga. In case of an effect on binding it is expected that the product distribution is different from the wild type enzyme however in case of involvement in catalysis only a slight effect, if any, on the product distribution is expected. In Fig. 5 two selected HPLC profiles are presented showing the product distribution when pga is hydrolyzed to the same extent by wild type PGII and mutated PGII. Apart from a small change in G1 contribution there is hardly any change in product distribution. A small effect on G1 is to be expected since the active site histidine is located at the junction of subsites -1 and + 1. These data demonstrate that His223 is involved in catalysis and not in binding.
Acknowledgement This work was financially supported by the European Community grant no. AIR2-CT941345.
References 1) Rexov/t-Benkov~i, L. (1973) Eur. J. Biochem. 39, 109-115. 2) Rexovfi-Benkov~t, L. and Marckov~i, M. (1978) Biochem. Biophys. Acta 523, 162-169. 3) Kester, H.C.M. and Visser, J. (1990) Biotechn. Appl. Biochem. 12, 150-160. 4) Bussink, H.J.S., Buxton, F.P., Fraaye, B.A., de Graaff, L.H. and Visser, J. (1992) Eur. J. Biochem. 208, 83-90. 5) Harmsen, J.A.M., Kusters-van Someren, M.A. and Visser, J. (1990) Curr. Genet. 18, 161-166. 6) Kusters-van Someren, M.A., Flipphy, M.J.A., De Graaff, L.H., van den Broek, H.C., Kester, H.C.M., Hinnen, A. and Visser, J. (1992). Mol. Gen. Genet. 234, 113-120. 7) Goosen, T., Bloemheuvel, G., Gysler, C., de Bie, D.A., van den Broek, H.W.J and Swart, K. (1987) Curr. Genet. 11,499-503. 8) Pontecorvo, G. Roper, J.A., Hemmons, L.J., MacDonald, K.D. and Bufton, A.W.J. (1953) Adv. Genet 5, 141-238. 9) Stephens, B.G., Felkel, H.J.Jr. and Spinelli, W.M. (1974) Anal. Chem. 692-696. 10) Thoma, J.A., Rao, G.V.K., Brothers, C. and Spradlin, J. (1971) J. Biol. Chem. 246, 5621-5635. 11) Hiromi, K., Nitta, Y., Numata, C. and Ono, S. (1973) Bioch. Biophys Acta 302, 362375. 12) Robyt, J.F. and French, D. (1967) Arch. Biochem. Biophys. 122, 8-16. 13) Robyt, J.F. and French, D. (1970) Arch. Biochem. Biophys. 138, 662-670. 14) Pasculli, R., Geraeds, C., Voragen, F and Pilnik, W. (1991) Lebensm. Wiss. u. Technol. 24, 63-70. 15) Schrtiter, K.-H., Arkema, A., Kester, H.C.M., Visser, J. and Dijkstra, B.W. (1994) J. Mol. Biol. 243,351-352.
Kinetics and mode of action of
221
AspergiUus niger polygalacturonases
Jacques A.E. Benen, Harry C.M. Kester, Lucie Parenicovfi and Jaap Visser. Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreyenlaan 2, 6703 HA Wageningen, The Netherlands.
Abstract Endo-polygalacturonases I and II (PGI and PGII) isolated from recombinant A. niger were characterized with respect to pH optimum, activity on polygalacturonic acid (pga), mode of action and kinetics on oligogalacturonates. Vmax and Km values using pga as a substrate at the optimum pH 4.1 were calculated as 500 U/mg and 0.15 mg/ml and 2000 U/mg and 0.15 mg/ml for PGI and PGII, respectively. Mode of action analysis revealed a random cleavage pattern for PGII while for PGI multiple attack on a single chain was observed. For PGII a partial subsite map was obtained. Site directed mutagenesis of His223 of PGII with subsequent analysis of the mutated PGII revealed that His223 is essential for catalysis.
Introduction Polygalacturonases from primarily fungal origin have been studied since several decades. Nowadays many genes encoding polygalacturonases, both exo- and endo-acting, from numerous different species have been cloned. Despite the large number of genes available and the long record of polygalacturonase studies most studies were directed at the purification of the enzymes and a limited characterization comprising mostly whether the enzyme is exo- or endo-acting, the activity on polygalacturonic acid and the pH, temperature and ionic strenght optima while very few studies were carried out toward the understanding of the mode of action, determined by the characteristics of the individual subsites of the enzymes. First studies addressing this problem via the determination of the number of subsites of an Aspergillus niger polygalacturonase and identification of catalytically important residues of this enzyme were described by Rexov~i-Benkovfi [1, 2]. Unfortunately, these studies have found only little follow up by other research groups [3]. Therefore detailed knowledge about 'subsite architecture' of these industrially important enzymes is scarce. Bussink et al. [4] and Kusters-van Someren et al. [5] have shown that in A. niger for both polygalacturonases (PGs) and pectin lyases (PLs) families of genes are present: seven
222 PG and six PL encoding genes were identified [4, 5]. The occurrence of families of genes encoding different enzymes raises the possibility of a concerted action of the enzymes of one or both families in the degradation of pectin. In order to clarify the role of the individual enzymes in the pectin degradation a comparative study in this respect was initiated. For this, individual genes were fused with the pkiA promoter of the glycolytic pyruvate kinase gene that allows expression of the individual genes under conditions where all other pectinases are repressed. Careful analysis of mode of action, kinetic parameters and subsite affinities of the enzymes on model and natural substrates will reveal the role of the individual enzymes in pectin degradation. Here we report on the characterization of recombinant PGI and PGII, the two most abundant PGs in the commercial Rapidase preparation [3] and of a site specific mutated PGII in which His223 was changed into Ala.
Materials and Methods Molecular biology. All DNA manipulations were performed using standard techniques. Promoter-gene fusions were constructed as described by Kusters-van Someren et al. [6]. Site directed mutagenesis of His223 of PGII was carried out in essentially the same way. PCR generated DNA fragments were checked for undesired mutations by sequence analysis. Transformation of A. niger NW228 (pyr, prtF) with appropriate plasmids was done as described before [7]. Growth and purification. A. niger strains transformed with either the pki-pgaI or pla'-pgalI fusion were grown in batch in 1L flasks containing 350 ml minimal medium according to Pontecorvo et al. [8] supplemented with Vishniacs spore element solution, 0 . 1 % yeast extract, 70 mM NH4C1 and 4 % fructose as a carbon source. Cultures were inoculated with 1 • 106 spores/ml and grown at 30 ~ in a rotary shaker for 18 hrs. Mycelium was separated from the medium by filtration over a nylon membrane. The filtrate was adjusted to pH 6.0 and loaded onto a DEAE-Sepharose Fast Flow column pre-equilibrated at pH 6.0 (10 mM Bis-Tris/HC1). Elution was performed with a linear gradient, 0-600 mM NaC1, in 10 mM Bis-Tris pH 6.0. SDS-PAGE of individual fractions demonstrated that the enzymes were pure after this separation. Fractions containing the enzyme were pooled and dialysed against 50 mM Na-acetate pH 4.5 and stored at either -20 ~ or 4 ~ until use. Mode of action and kinetics. Routine polygalacturonase assays were performed in a reaction mixture containing 50 mM Na-acetate pH 4.2 and 0.25 % w/v pga at 30 ~ The release of reducing sugars was determined according to Stephens et al. [9]. For determination of pH optima the 50 mM Na-acetate buffer was replaced by Mcllvain buffers. For the determination of kinetic parameters and for the mode of action of the enzymes reaction products were analysed on a Dionex BioLC/high-performance chromatography system using a Carbo Pac PA-100 anion-exchange column (25 cm x 4 mm) with pulsed amperometric detection. The samples loaded were eluted with a linear gradient of 0.15-0.60 M Na-acetate in 0.1 M NaOH at 1 ml/min in 22 min. Products were quantitated via calibration mixes containing oligogalacturonates with DP 1-8 (G1-G8) at 0.1 mM each and via an internal standard of 0.1 mM glucuronic acid (eluting between G1 and G2) with 50 pl injections.
223
Results and Discussion The PGI and PGII produced from strains transformed with the promoter gene fusion are in all respects tested identical to those enzymes obtained from the wild type strain when grown on pectic substances. For both PGI and PGII the pH optimum is 4.1-4.2 in 50 mM Na-acetate buffer, 30 ~ All further kinetic analyses were performed under these conditions. PGII. Using pga as a substrate Km and Vmax of PGII were calculated as 0.15 mg/ml and 2050 U/mg, respectively. The high Vmax and low Km demonstrate that pga is a good substrate for PGII. By following the product formation as a function of time (Fig. 1) it was demonstrated that PGII is a randomly cleaving endo polygalacturonase. The progression of substrates is characterised by an initial transient increase of higher oligogalacturonates which are gradually converted to oligomers with lower DP, eventually resulting in a mixture of G1, G2 and G3. The rather strong transient increase of G4 and G5 is not a result of transglygosylation, since PGII is an inverting enzyme (see Biely et al. elsewhere in this volume), but is merely due to the slow hydrolysis of these compounds as will be discussed below.
G2
0.4
G3
G1 IS
G4
0.3 90 min
~- 0.2 _
-
-
-
35 min
0.1
J A
0.0 0
5
~ _ . _ . _ . ~ _ A
10
15
10 min I 20
minutes
Figure 1. HPLC analysis of product progression during hydrolysis of 0.25 % polygalacturonate by PGII. Aliquots were withdrawn from the reaction mixture at timed intervals and reactions were stopped by raising the pH of the sample to pH 8.0 by mixing with 1 volume 25 mM Na-phosphate pH 9.5. G1 to G5 indicate the oligogalacturonates with corresponding degree of polymerization. The vertical axis shows the responce of the pulsed amperometric detector and the horizontal axis the elution time. Times of sampling are indicated above the trace.
224
Table I. Mode of action of PGII. Bond cleavage frequencies (BCF) in percentage for oligogalacturonates. The reducing or end of the products is indicated with a solid circle. The position of cleavage is indicated with a solid triangle. DP
BCF (%)
4 5
6
o
o
o o
Products
o
o
o
9
100
o o
o o
o o
9 o
9
67 33
dimer
o o o
o o o
o o o
9 o o
9 o
35 57 8
dimer trimer
9
A
In order to estimate the number of subsites, the binding affinities, the location of the active site and the cleavage patterns reduced and non reduced oligogalacturonates of DP 4 to 6 were used as substrates and the resulting products analysed by HPLC and TLC. Table I lists the bond cleavage frequencies for PGII. G4 is exclusively split in 1-3 mode while reduced G4 was not hydrolysed. G5 is cleaved in the 1-4 and 2-3 mode at 67 % and 33 % respectively, while reduced G5 is only split into reduced G2 and G3. The reduced G6 is not cleaved in the 1-5 mode, while reduced G2 and reduced G3 are readily formed. The non-reduced G6 is cleaved in 1-5, 2-4 and 3-3 modes yielding equimolar product pairs as was also seen for the cleavage of G4 and G5. These data demonstrate that cleavage of the glycosidic bond occurs from the reducing end. In time course experiments using different oligogalacturonates at several concentrations the turnover numbers and Km values were estimated for each oligomer in the individual binding modes and used for the calculation of the thermodynamic parameters of PGII according to Thoma et al. [10] and Hiromi et al. [11]. In Table II the data are presented. An approximation of the intrinsic rate constant, kint, was calculated from Vmax on pga (kint = kcat). From kint and Km and the turnover number for each binding mode the binding energies for each mode were calculated which were in turn used for the calculation of the individual subsite affinities listed in Table III. Since G4 is the smallest substrate used in this study, it is not possible to obtain information of the subsites at positions -3 to 1.
225 Table II, Kinetic and thermodynamic parameters of PGII using oligogalacturonates as substrates. Kp was calculated from Kp=[ko/Km]/kint while kint was obtained as described in the text. AG was calculated from -AGp=RTlnKp + 10 kJ/mole. Mode indicates the cleavage mode DP
Mode
Km • 10-6 (M)
ko
ko/Km x 10-6 Kp
(s -1)
(M -1 s-1)
(M -1)
AGp (kJ/mole)
4
G3 + G1 22
103
4.7
8048
-22.6
5
G4 + G1 13 G3 + G2 25
315 159
24.2 6.4
41438 10960
-26.7 -23.4
G5 + G1 40 G4 + G2 16 G3 + G3 31
148 386 56.5
3.7 24.1 1.8
6336 41267 3082
-22.0 -26.7 -20.2
Table III. Subsite affinities Ai for PGII (i denotes the subsite number). Subsites with a '-' prefix are located towards the non-reducing end of the substrate while subsites with a ' + ' prefix are located towards the reducing end. The active site is located between subsites -1 and + 1. Subsites -3 to + 1 were determined as one value. Subsite (Ai) Affinity (kJ/mole)
-4 +4.2
-3 / + 1 +22.6
+2 +0.8
The subsite map and the data on reduced oligomers, which showed formation of reduced G2 on reduced G5, indicate that the number of subsites is 5, stretching from position-4 to 1. The sum of the individual turnover numbers for each oligomer also shows that the rate of hydrolysis of the oligomers with DP4 and DP5 is much slower than of those with higher DP which is reflected in the product progression curves in Fig. 1. Rexov~i-Benkov~i [1] studied an A. niger endopolygalacturonase which might have been the same as the PGII described here; the cleavage pattern described is very much the same as found here and the pH optimum is exactly the same. The number of subsites for that enzyme was found to be four which is not in agreement with the number found for PGII. However, bearing in mind that in the study of Rexovgt-Benkovgt the individual binding modes of each oligogalacturonate were not addressed and therefore no subsite map was obtained the additional fifth subsite might have been overlooked.
226 PGI.
In a similar way as for PGII the kinetics for PGI were addressed. Using pga as a substrate Km and Vmax were 0.15 mg/ml and 500 U/mg, respectively. Fig. 2 shows the product progression upon pga hydrolysis. There is a strong increase of G1, G2 and G3 which is accompanied by a transient increase of G4 and G5 while there is only a small increase of oligomers with DP higher than 5. The transient increase of G4 and G5 is not due to transglycosylation since like PGII, PGI is also an inverting enzyme (see Biely et al. elsewhere in this volume). The profiles of PGI suggest that the enzyme after first random cleavage of the polymer substrate degrades the higher oligomers formed preferentially via hydrolysis of terminal residues at the reducing end. The latter was demonstrated by hydrolysis of reduced oligogalacturonates while the former was investigated in more detail by analysing the mode of action and cleavage rates on defined oligomers ranging from DP 4 to 8 . PGI hydrolyses G4 mainly to G1 and G3 at equimolar amounts as expected and a small amount is digested into G2 (Results not shown). G5 is cleaved to G1 and G4 in again equimolar amounts and to almost the same extent to G2 and G3 also in equimolar amounts (Results not shown). Thus, for G4 and G5, PGI is not much different from PGII, only the bond cleavage frequencies being slightly different. Upon hydrolysis of G6 to G8 the formation of equimolar product pairs is not observed anymore. With G6 as substrate G1 is formed at least twice as fast as G5 while G4 is formed
IS G2
0.14 0.12 0.10 0
0.08
G3 G4 G5
0.06 0.04
J
0.02
IS
0.00 0
G2 G3 G4 G5 I
I
10
15
10 min I 20
minutes
Figure 2. HPLC analysis of product progression during hydrolysis of 0.25 % polygalacturonate by PGI. Aliquots were withdrawn from the reaction mixture at timed intervals and reactions were stopped by raising the pH of the sample to pH 8.0 by mixing with 1 volume 25 mM Na-phosphate pH 9.5. G1 to G5 indicate the oligogalacturonates with corresponding degree of polymerization. The vertical axis shows the responce of the pulsed amperometric detector and the horizontal axis the elution time. Times of sampling are indicated above the trace.
227 faster as G2 (Fig 3). A similar behavior was observed by Robyt and French [12, 13] for cxamylase. They demonstrated that this type of product ratios, that differ from the beginning of the reaction, are due to multiple attack on a single chain. A ratio-plot according to Robyt and French [13] for G1 and G5 formation from G6 supported that PGI exibits multiple attack on G6 (not shown). The fact that still G5 is formed upon hydrolysis of G6 demonstrates that not all G6 bound in the G5-G1 mode is cleaved in a repetitive way. Thus, only a fraction of the substrate bound in this mode is subject to repetitive attack. The ratio between cleavage in this way and normal cleavage into G1 and G5 is determined by the dissociation constant of the G5 generated upon cleavage and the first order rate constant that is responsible for the shift of the bound G5 into the G4-G1 mode. Since still a considerable amount of G5 is formed, the dissociation constant of G5 and the first order 'shift' rate constant are of the same magnitude. However, from G7 and G8 as substrates it is clear that the ratio between the dissociation constant and 'shift' rate constant is completely in favor of shift when DP equals 6 or 7. With G7 as a substrate quite large amounts of G1 are formed while there is no detectable formation of the corresponding G6, only a rapid accumulation of G5 is observed. Similarly with G8 as a substrate quite large amounts of G1 and G2 are formed while there is no detectable formation of the corresponding G7 and G6, only again a rapid accumulation of G5 occurred which indicates that the multiple attack ceases when DP is down to 5 or 4, hence when dissociation is favored over shift. The previous data allow a clear interpretation of Fig. 2. PGI initially hydrolyses pga in a random endolytic way generating higher oligomers which is accompanied by rapid multiple attack of the higher oligomers in an exolytic way to yield mainly monomers and dimers from the reducing end. Therefore no transient accumulation of higher oligomers takes place. The appearance of transient G5 and G4 is due to the fact that the multiple attack of the higher oligomers stops when DP is 4 or 5 and these product are released. The transient accumulation of the tetramer partly originates from the particular binding mode of the higher oligomers. At present we are working on a kinetic model that describes the action pattern of PGI. It has been suggested that the multiple or single attack can be distinguished based on the relative rate of accumulation of mono- di- and trimers upon hydrolysis of polymeric substrate. At first glance this appears to be valid when comparing for example the mode of action of PGI and PGII (see Fig. 1 and Fig. 2). In a comparative study of three polygalacturonases by Pasculli et al [14] the enzymes were classified according these criteria while the rate of accumulation was by far not as clearcut differing as found for PGI and PGII. The observed profiles in that study can also be explained by assuming different rates of hydrolysis of the smaller (DP 4-6) oligogalacturonates. If for instance the rate of hydrolysis of G4 and G5 of PGII would have been higher then the progression curves would have more resembled those of PGI. The only way to discern between multiple or single attack is by analysis of the stochiometry of product pairs upon hydrolysis of oligomeric substrates at initial stages of reaction.
228 40 gl ,.i-,
o
(D c
30
.,.-~
(/) 0
20
g5
E 0c -
~c -
g4 g2 g3
10
~n-'~ 0
T 5
I I0
I 15
I 20
I 25
I 30
minutes
Figure 3. HPLC analysis of product progression during hydrolysis of 0.5 mM hexagalacturonate by PGI. Aliquots were withdrawn from the reaction mixture at timed intervals and reactions were stopped by raising the pH of the sample to pH 8.0 by mixing with 1 volume 25 mM Na-phosphate pH 9.5. G1 to G5 indicate the oligogalacturonates with corresponding degree of polymerization.
PGII His223Ala. Apart from mode of action and kinetics of wild type enzymes structure function relationships of these industrially important enzymes is of high interest to provide the necessary knowledge for genetic engineering of desired properties. As a first approach the identification of catalytically important residues was addressed in conjunction with the elucidation of the three dimensional structure [15]. Rexovfi-Benkov~i and Marckov~t [2] obtained evidence for the possible involvement of a histidine in catalysis of a fungal endopolygalacturonase. The same results were obtained for PGII using diethyl pyro-carbonate as a modifying agent in the presence or absence of substrate (Kester, unpublished). However chemical modification studies can never give conclusive evidence for the participation of the modified residue in catalysis or binding. The method to dissect the role of individual residues is site directed mutagenesis. Upon alignment of 25 endo- and exo-polygalacturonases taken from the swiss.prot. database only one histidine appeared to be conserved throughout. The conserved histidine at position 223 in PGII was changed into alanine, a small apolar residue. The mutation has a dramatic effect on Vmax using pga as substrate which decreases from 2050 U/mg to 10 U/mg; the Km did not change significantly, however. The mutation also affected the pH optimum of the enzyme as is shown in Fig. 4. The pH optimum narrows down to one pH unit. Since the polygalacturonate concentration used is well above the Km the apparent velocities can roughly be regarded as Vmax values at each pH. So, the fall and rise of the activity within one pH unit strongly suggests that catalysis is governed by only one ionisable group in the mutated enzyme, a glutamate or an aspartate. Thus, the
229
100 >,
80
60
~
9"~ rr
40 20 0 I
I
I
I
2.5
3
3.5
4
I
I
I
I
4.5
5
5.5
6
pH
Figure 4. Relative activities of wild type PGII and His223Ala mutated enzyme as a function of pH. Wild type enzyme, solid circles; His223Ala mutated enzyme, open circles. 0.30 0.25
' -
IIs
gl.
g3
0.20
G 0:=L
0.15 IS 0.10
g2
0.05
j~
0.00
I
0
5
i
I
,
10
!
!
15
20
minutes
Figure 5. Selected HPLC elution profile of products obtained after incubation of 0.25% polygalacturonate with PGII, upper trace, and PGII H223A, lower trace, respectively, demonstrating the effect of the mutation on catalysis. G1 to G3 indicate the peaks of the corresponding oligogalacturonates. IS indicates the internal standard, glucuronate. The vertical axis shows the pulsed amperometric detector response while the horizontal axis shows the retention time.
230 strong effect on Vmax, the absence of an effect on Km and the striking effect on the pH optimum, the shift in the direction expected, provide evidence for the involvement of His223 in catalysis rather than binding. This is again affirmed by the analysis of the products formed after hydrolysis of pga. In case of an effect on binding it is expected that the product distribution is different from the wild type enzyme however in case of involvement in catalysis only a slight effect, if any, on the product distribution is expected. In Fig. 5 two selected HPLC profiles are presented showing the product distribution when pga is hydrolyzed to the same extent by wild type PGII and mutated PGII. Apart from a small change in G1 contribution there is hardly any change in product distribution. A small effect on G1 is to be expected since the active site histidine is located at the junction of subsites -1 and + 1. These data demonstrate that His223 is involved in catalysis and not in binding.
Acknowledgement This work was financially supported by the European Community grant no. AIR2-CT941345.
References 1) Rexov/t-Benkov~i, L. (1973) Eur. J. Biochem. 39, 109-115. 2) Rexovfi-Benkov~t, L. and Marckov~i, M. (1978) Biochem. Biophys. Acta 523, 162-169. 3) Kester, H.C.M. and Visser, J. (1990) Biotechn. Appl. Biochem. 12, 150-160. 4) Bussink, H.J.S., Buxton, F.P., Fraaye, B.A., de Graaff, L.H. and Visser, J. (1992) Eur. J. Biochem. 208, 83-90. 5) Harmsen, J.A.M., Kusters-van Someren, M.A. and Visser, J. (1990) Curr. Genet. 18, 161-166. 6) Kusters-van Someren, M.A., Flipphy, M.J.A., De Graaff, L.H., van den Broek, H.C., Kester, H.C.M., Hinnen, A. and Visser, J. (1992). Mol. Gen. Genet. 234, 113-120. 7) Goosen, T., Bloemheuvel, G., Gysler, C., de Bie, D.A., van den Broek, H.W.J and Swart, K. (1987) Curr. Genet. 11,499-503. 8) Pontecorvo, G. Roper, J.A., Hemmons, L.J., MacDonald, K.D. and Bufton, A.W.J. (1953) Adv. Genet 5, 141-238. 9) Stephens, B.G., Felkel, H.J.Jr. and Spinelli, W.M. (1974) Anal. Chem. 692-696. 10) Thoma, J.A., Rao, G.V.K., Brothers, C. and Spradlin, J. (1971) J. Biol. Chem. 246, 5621-5635. 11) Hiromi, K., Nitta, Y., Numata, C. and Ono, S. (1973) Bioch. Biophys Acta 302, 362375. 12) Robyt, J.F. and French, D. (1967) Arch. Biochem. Biophys. 122, 8-16. 13) Robyt, J.F. and French, D. (1970) Arch. Biochem. Biophys. 138, 662-670. 14) Pasculli, R., Geraeds, C., Voragen, F and Pilnik, W. (1991) Lebensm. Wiss. u. Technol. 24, 63-70. 15) Schrtiter, K.-H., Arkema, A., Kester, H.C.M., Visser, J. and Dijkstra, B.W. (1994) J. Mol. Biol. 243,351-352.