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Control of Mung Bean Pectinmethylesterase Isoform Activities

2001; Elsevier BV; Volume: 276; Issue: 12 Linguagem: Inglês

10.1074/jbc.m001791200

1083-351X

Renée Goldberg, Monique Pierron, Marianne Bordenave, Christelle Breton, Claudine Morvan, C. Hervé Du Penhoat,

Biofuel production and bioconversion

Well-characterized pectin samples with a wide range of degrees of esterification (39–74%) were incubated with the solubilized pure α and γ isoforms of pectinmethylesterase, from mung bean hypocotyl (Vigna radiata). Enzyme activity was determined at regular intervals along the deesterification pathway at pH 5.6 and pH 7.6. It has been demonstrated that the distribution of the carboxyl units along the pectin backbone controls the activity of the cell wall pectinmethylesterases to a much greater extent than the methylation degree, with a random distribution leading to the strongest activity. Polygalacturonic acid was shown to be a competitive inhibitor of the α isoform activity at pH 5.6 and to inhibit the γ isoform activity at both pH 5.6 and pH 7.6. Under these conditions, the drop in enzyme activity was shown to be correlated to the formation of deesterified blocks of 19 ± 1 galacturonic acid residues through simulations of the enzymatic digestion according to the mechanisms established previously (Catoire, L., Pierron, M., Morvan, C., Herve du Penhoat, C., and Goldberg, R. (1998) J. Biol. Chem.273, 33150–33156). However, even in the absence of inhibition by the reaction product, activity dropped to negligible levels long before the substrate had been totally deesterified. Comparison of α and γ isoform cDNAs suggests that the N-terminal region of catalytic domains might explain their subtle differences in activity revealed in this study. The role of pectinmethylesterase in the cell wall stiffening process along the growth gradient is discussed. Well-characterized pectin samples with a wide range of degrees of esterification (39–74%) were incubated with the solubilized pure α and γ isoforms of pectinmethylesterase, from mung bean hypocotyl (Vigna radiata). Enzyme activity was determined at regular intervals along the deesterification pathway at pH 5.6 and pH 7.6. It has been demonstrated that the distribution of the carboxyl units along the pectin backbone controls the activity of the cell wall pectinmethylesterases to a much greater extent than the methylation degree, with a random distribution leading to the strongest activity. Polygalacturonic acid was shown to be a competitive inhibitor of the α isoform activity at pH 5.6 and to inhibit the γ isoform activity at both pH 5.6 and pH 7.6. Under these conditions, the drop in enzyme activity was shown to be correlated to the formation of deesterified blocks of 19 ± 1 galacturonic acid residues through simulations of the enzymatic digestion according to the mechanisms established previously (Catoire, L., Pierron, M., Morvan, C., Herve du Penhoat, C., and Goldberg, R. (1998) J. Biol. Chem.273, 33150–33156). However, even in the absence of inhibition by the reaction product, activity dropped to negligible levels long before the substrate had been totally deesterified. Comparison of α and γ isoform cDNAs suggests that the N-terminal region of catalytic domains might explain their subtle differences in activity revealed in this study. The role of pectinmethylesterase in the cell wall stiffening process along the growth gradient is discussed. pectinmethylesterase degree of methylesterification multiple attack mechanism polygalacturonic acid single-chain mechanism polymerase chain reaction Pectinmethylesterases (PMEs)1 are cell wall-bound proteins present in almost all plants and phytopathogenic microorganisms. They modify pectic homogalacturonan chains, generating free carboxyl groups along the polygalacturonan backbone and releasing protons into the apoplasm. Their action can therefore lead to antinomical effects, especially in primary cell walls. On one hand, the pH decrease should enhance expansin activity and in turn increase the cell wall extensibility (1McQueen-Mason S. Durachko D.M. Cosgrove D. Plant Cell. 1992; 4: 1425-1433Crossref PubMed Scopus (746) Google Scholar), but, on the other hand, the generation of carboxyl blocks would be expected to allow the formation of multichain structures via calcium bridges (2Goldberg R. Morvan C. Jarvis M.C. Visser J. Voragen A.G.J. Pectins and Pectinases. Elsevier Science Publishers B. V., Amsterdam1996: 151-172Google Scholar). Such structures would greatly affect the physical properties of pectin, due to the assembly of pectic chains into expanded, highly hydrated gel networks decreasing cell wall porosity and also cell wall extensibility (3Carpita N.C. Gibeaut D.M. Plant J. 1993; 3: 1-30Crossref PubMed Scopus (2852) Google Scholar). Such Ca2+bridging requires the occurrence of nearly 10 successive carboxyl groups (4Van Cutsem P. Messiaen J. Visser J. Voragen A.G.J. Pectins and Pectinases. 17. Elsevier Science Publishers B. V., Amsterdam1996: 135-149Google Scholar) and implies that the PMEs work processively along the galacturonan chain. Unfortunately, although PME biochemical and molecular characteristics have been widely investigated (5Bordenave M. Linskens H.F. Jackson J.F. Modern Methods of Plant Analysis. 17. Springer-Verlag, New York, Berlin1996: 165-180Google Scholar, 6Albani D. Altostair I. Arnison P.G. Fabijanski S.F. Plant Mol. Biol. 1991; 16: 501-513Crossref PubMed Scopus (115) Google Scholar, 7Mu H. Stains J.P. Kao T. Plant Mol. Biol. 1994; 25: 539-544Crossref PubMed Scopus (75) Google Scholar, 8Richard L. Qin L.X. Goldberg R. Gene ( Amst. ). 1996; 170: 207-211Crossref PubMed Scopus (28) Google Scholar), a limited number of studies of their action pattern have been reported (9de Vries J.A. Rombouts F.M. Voragen A.G.J. Pilnik W. Carbohydr. Polym. 1983; 3: 245-258Crossref Scopus (52) Google Scholar, 10Versteeg C. Pectinesterases from the Orange Fruit: Their Purification; General Characteristics, and Juice Cloud-destabilizing Properties. Ph. D. thesis. Agricultural University of Wageningen, The Netherlands1979Google Scholar, 11Markovic O. Kohn R. Experientia ( Basel ). 1984; 40: 842-843Crossref Scopus (43) Google Scholar, 12Andersen A.K. Larsen B. Grasladen H. Carbohydr. Res. 1995; 273: 93-98Crossref Scopus (35) Google Scholar, 13Grasladen H. Andersen A.K. Larsen B. Carbohydr. Res. 1996; 289: 105-114Crossref Scopus (72) Google Scholar). Recently, using partially depolymerized pectin samples and different PME isoforms extracted from mung bean hypocotyl cell walls, we (14Catoire L. Pierron M. Morvan C. Herve du Penhoat C. Goldberg R. J. Biol. Chem. 1998; 273: 33150-33156Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) determined both enzyme activity and average product structure at regular intervals along the deesterification pathway. Simulations of different mechanisms by fitting to the experimental data provided information on the possible action patterns of three isoforms. In the case of two of them, the so-called PEα (pI around 7.5) and PEγ (pI above 9) isoforms, a single-chain mechanism (SCM) associated with a free carboxyl group at the second nearest neighbor position was postulated at pH 5.6, whereas some multiple attack mechanism (MAM) was required to reproduce the experimental data at pH 7.6. Although these action patterns reproduced the free carboxyl and methylester distribution in pectin incubated with PEα and PEγ at both pHs for the entire degree of esterification (DE) range observed experimentally, they did not explain the marked decrease in activity for DE values below 50–60%. Indeed, in the simulated digestions, deesterification occurred smoothly down to DE values between 2–4%. In the present study, we set out to further investigate the effects of neutral and acidic pHs on the development of the deesterification process catalyzed by the neutral and alkaline PME isoforms (PEα and PEγ, respectively). We wanted to determine what limits the deesterification process in situ because, in young cell walls, active PMEs coexist with highly methylated pectins. Three well-characterized commercial pectin samples differing in their DE were chosen for these investigations. We endeavored (a) to determine the kinetic constants of deesterification, (b) to establish the activity profiles as a function of the deesterification pathway for all three samples, (c) to check both experimentally and with simulations possible limiting factors such as inhibition by the acidic blocks produced during the reaction, (d) to evaluate the lengths of such acidic blocks through simulations, and (e) to compare the cDNA-deduced peptide sequences of both isoforms to understand the possible interactions required for the stabilization of pectin-PME binding at pH 5.6 and pH 7.6. The information obtained with this approach was expected to shed light on the role of the different PME isoforms in the cell wall stiffening process that occurs along the mung bean hypocotyl. The three pectin samples PS1, PS2, and PS3, which differed in their DE, were graciously supplied by Herbstreith & Fox KG (Neuenbürg, Germany). Cell walls were isolated from the upper 2.5 cm of hypocotyl tissues of 3-day-old seedlings of mung bean (Vigna radiata L. Wilzeck) according to a procedure described previously (15Goldberg R. Morvan C. Roland J.C. Plant Cell Physiol. 1986; 27: 523-532Google Scholar). PMEs bound to the cell wall fragments were solubilized with 1 m NaCl, and the different isoforms were recovered as described by Bordenave and Goldberg (16Bordenave M. Goldberg R. Phytochemistry. 1993; 33: 999-1003Crossref Scopus (34) Google Scholar, 17Bordenave M. Goldberg R. Plant Physiol. 1994; 106: 1151-1156Crossref PubMed Scopus (46) Google Scholar). PME activity was measured titrimetically by following the release of protons from the pectin sample in the presence of 150 mm NaCl (total volume of the assay, 6 ml). The protons were titrated with 10 mm NaOH under nitrogen, the pH was maintained at pH 5.6 or pH 7.6 with an automatic titrator (TTT 80; Radiometer), and the reaction rate was expressed as μeq H+ released/min. The reaction was stopped by lowering the pH to pH 3 with 0.05 nHCl. To check whether some differences could be detected in pectin behavior after action of PEα at either pH 5.6 or pH 7.6, interaction experiments (18Penel C. Van Custem P. Greppin H. Phytochemistry. 1999; 51: 193-198Crossref Scopus (29) Google Scholar) were run as follows. Pectins with a DE of 74% were treated with PEα as described above until their DE reached 50%, and then HCl was added rapidly to shift the pH value to pH 3, inactivate the enzyme, and facilitate the precipitation of pectins with ethanol. After several cycles of precipitation/solubilization in deionized water, pectins (cleansed of salts) were finally dissolved in water at a concentration of 10 mg/ml, corresponding to about 2.5 mm free galacturonic acid. Increasing amounts of calcium chloride were added to the solutions, and pectins were allowed to precipitate. To evaluate the interaction of pectins with calcium ions, the amount of residual sugars was determined colorimetrically in the supernatant after centrifugation at 10,000 × g for 15 min, according to the method of Dubois et al. (19Dubois M. Gille K.A. Hamilton J.K. Reberts P.A. Smith F. Anal. Chem. 1956; 28: 350-356Crossref Scopus (40233) Google Scholar). Deesterification simulations were conducted with in-house software as described previously (14Catoire L. Pierron M. Morvan C. Herve du Penhoat C. Goldberg R. J. Biol. Chem. 1998; 273: 33150-33156Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Starting samples consisting of 1,000 chains of 100 residues with a Bernoullian (random) distribution of methylester groups and DE values of 73% (PS1), 53% (PS2), and 38% (PS3) were constructed with a random number generator (E, methylesterified galacturonic acid residue; U, galacturonic acid residue with a free carboxyl group; X, unspecified residue that may be E or U; E, residue in the process of deesterification). PME deesterification was simulated as random attack followed by blockwise deesterification according to the SCM or MAM that included constraint criteria. The latter feature took into account the requirement that a free carboxyl group (U) be located 2 residues away from the point of attack (UXE constraint). At regular intervals along the deesterification pathway (DE steps of 5%), average populations such as the number of blocks of various lengths were determined. To assess the influence of small variations in DE (∼1–2%) on the block size of the largest U block, data were also simulated five times with different initial seeds at DE intervals of 1% for the SCM with the UXE constraint. Simulations were repeated at least five times with different initial seeds for the random number generator, and the average values for the U block populations for the single-chain and multiple attack mechanisms were determined. PCR was used to amplify the coding region of PEα using degenerate primers that were designed with regard to internal peptides of the protein, as described previously (20Bordenave M. Breton C. Goldberg R. Huet J.-C. Perez S. Pernollet J.-C. Plant Mol. Biol. 1996; 31: 1039-1049Crossref PubMed Scopus (26) Google Scholar). Total RNA was prepared from 3-day-old mung bean seedlings according to the guanidinium thiocyanate method (21Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63087) Google Scholar) and used to synthesize cDNA (first-strand cDNA synthesis kit; Amersham Pharmacia Biotech). Using primers prPEW (sense 5′-AGGNGCNTAYTTYGARAA) and prPEZ (antisense 5′-ANCKNCCYTGNGCNGTRAA), which code for the peptide sequences GAYFEN and FTAQGR of two internal peptides, respectively, a 450-base pair fragment was obtained using cDNA as template. PCR was performed using Taq DNA polymerase (Appligene) with the following temperature profile: 1 min at 94 °C, 1 min at 50 °C, and 2 min at 70 °C, for 35 cycles. The purified PCR fragment was cloned into pMosBlue vector (Amersham Pharmacia Biotech). Upon sequencing, this fragment was shown to include the coding regions of several internal peptides. The 3′-/5′-rapid amplification of cDNA ends system (Life Technologies, Inc.) was then used to obtain the full-length cDNA of PMEα. For each cloned PCR product, three independent clones were sequenced on both strands using the T7 Sequenase Quick denature plasmid sequencing kit (Amersham Pharmacia Biotech). A cDNA library was also constructed in λZAP II (Stratagene) from polyadenylated RNA of mung bean hypocotyls using XL1-Blue cells as indicated by the manufacturer. The PCR fragment was labeled with digoxigenin (Roche Molecular Biochemicals) and used to screen the cDNA library. The pectin samples used in the assays have been described previously (22Catoire L. Goldberg R. Pierron M. Morvan C. Herve du Penhoat C. Eur. Biophys. J. 1998; 27: 127-136Crossref PubMed Scopus (54) Google Scholar). The three samples (PS1, PS2, and PS3) had average molecular masses of 188, 148, and 154 kDa and DEs of 74%, 54%, and 39%, respectively. These pectins presented some side chains (9%, 5%, and 5%, respectively), and the methoxyl distribution of PS1 and PS2 was random, whereas that of PS3 was slightly blockwise. This latter information could be reliably obtained from 13C NMR spectra because it was possible to establish triad populations (i.e.the relative fractions of EEE, EEU+UEE, and UEU trimers within the polymer could be evaluated by integration of the C6 signals). The percentages of methylester groups suitable for PME binding in PS1, PS2, and PS3 (i.e. satisfying the UXE constraint) were estimated to be 27%, 46%, and 60%, respectively, from the UXE populations of the theoretical pectins. The kinetic parameters of the two PME isoforms, PEα and PEγ, acting on various pectin samples differing in their methylesterification degrees have been collected in TableI. With both enzyme fractions and at pH 5.6 as well as at pH 7.6, the lower the DE, the higher the affinity. When the Km was calculated for the esterified residues suitable for enzyme binding, i.e. the esterified residues satisfying the requirement that a free carboxyl group be located at the second nearest neighbor position (UXEconstraint), the affinity also increased when the DE decreased. However, these increases were smaller than those calculated for the total methylester populations. With regard to the effects of pH, with the neutral isoform, PEα, whose activity is known to be modified relatively little by the ionic conditions (pH or saline concentration (20Bordenave M. Breton C. Goldberg R. Huet J.-C. Perez S. Pernollet J.-C. Plant Mol. Biol. 1996; 31: 1039-1049Crossref PubMed Scopus (26) Google Scholar)), the kinetic parameters (Vmax andKm) were rather similar at pH 5.6 and pH 7.6. In contrast, for PEγ, both the affinity and the maximal velocity were higher at pH 5.6, whatever the DE of the substrate.Table IKinetic parameters of PEα and PEγPectin samplepHPEαPEγKmVmaxKmVmax(1)(2)(1)(2)PS15.61.850.500.352.350.630.777.61.970.530.434.851.310.62PS25.60.710.330.470.340.161.027.60.480.220.420.520.240.62PS35.60.210.190.410.140.081.107.60.310.190.420.310.190.68The kinetic parameters (Km andVmax) of PEα and PEγ measured at pH 5.6 and pH 7.6 in 150 mm NaCl are given for various pectin samples: PS1, DE = 74%; PS2, DE = 54%; PS3, DE = 39%.Vmax is given as μmol of H+ min/μg, andKm is given as mm methylester groups, taking into account either the total concentration of methylester groups (1McQueen-Mason S. Durachko D.M. Cosgrove D. Plant Cell. 1992; 4: 1425-1433Crossref PubMed Scopus (746) Google Scholar) or the concentration of methylester groups suitable for PME binding (2Goldberg R. Morvan C. Jarvis M.C. Visser J. Voragen A.G.J. Pectins and Pectinases. Elsevier Science Publishers B. V., Amsterdam1996: 151-172Google Scholar), estimated as described under "Material and Methods." Open table in a new tab The kinetic parameters (Km andVmax) of PEα and PEγ measured at pH 5.6 and pH 7.6 in 150 mm NaCl are given for various pectin samples: PS1, DE = 74%; PS2, DE = 54%; PS3, DE = 39%.Vmax is given as μmol of H+ min/μg, andKm is given as mm methylester groups, taking into account either the total concentration of methylester groups (1McQueen-Mason S. Durachko D.M. Cosgrove D. Plant Cell. 1992; 4: 1425-1433Crossref PubMed Scopus (746) Google Scholar) or the concentration of methylester groups suitable for PME binding (2Goldberg R. Morvan C. Jarvis M.C. Visser J. Voragen A.G.J. Pectins and Pectinases. Elsevier Science Publishers B. V., Amsterdam1996: 151-172Google Scholar), estimated as described under "Material and Methods." Preliminary data indicate that upon incubation with PEα, pectins formed a gel that can be pelleted only at CaCl2 concentrations higher than 0.5 mm. Moreover, in the presence of 2.5 mm or more calcium ions, 80 ± 3% of the pectins sedimented at pH 5.6 and only 72 ± 4% of the pectins sedimented at pH 7.6. These results are in agreement with those of Penel et al. (18Penel C. Van Custem P. Greppin H. Phytochemistry. 1999; 51: 193-198Crossref Scopus (29) Google Scholar), who conducted similar experiments with polygalacturonic acid (PGA). The differences, although small, were observed repeatedly, whatever the concentration of CaCl2 (from 0.5 to 5 mm), indicating a difference in the distribution of the negative charges produced by the enzyme. To visualize the effects of DE on the time course of the reaction rate of the deesterification process, plots of activityversus DE have been traced for pectin samples differing in their DE (Fig. 1). Incubations were performed either at pH 5.6 or pH 7.6. The plots obtained with the two isoforms differed, but whatever the incubation conditions, none of the enzymatic fractions were able to generate totally deesterified pectins, and the final DE depended on the nature of the substrate. At pH 7.6, with the neutral isoform PEα, two successive phases were observed (Fig. 1 A): during the first phase, the reaction rate was nearly constant, whereas during the second one, it decreased rapidly. The DE value that corresponds to the greatest change in slope of the activity profiles is referred to as the slope change point (i.e. SCPEα or SCPEγ in TablesII and III) and is indicated with an arrow in Fig. 1. At pH 5.6, this second phase predominated because the activity dropped very rapidly. The plateau observed at pH 7.6 was much shorter with PEγ and decreased with DE (Fig. 1 B). With both isoforms, for a given DE, the reaction rate depended strictly on the nature of the pectin substrate. The reaction rate was always much lower when the DE resulted from enzymatic deesterification (e.g. the activity of PEα with PS1 at a DE of 53% as compared with that with PS2 at the beginning of the reaction). According to previous observations (14Catoire L. Pierron M. Morvan C. Herve du Penhoat C. Goldberg R. J. Biol. Chem. 1998; 273: 33150-33156Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), both isoforms produce acidic blocks along the pectin chains at a rate that depends on the pH. In contrast, the native pectin samples are characterized by a random distribution of the acidic units (22Catoire L. Goldberg R. Pierron M. Morvan C. Herve du Penhoat C. Eur. Biophys. J. 1998; 27: 127-136Crossref PubMed Scopus (54) Google Scholar). These data indicate that the distribution of the carboxyl and methoxyl groups along the polymer backbone is an important factor for the activity, with a random distribution of the carboxyl units inducing in all cases a higher activity than a blockwise one.Table IIDistribution of U blocks during digestion by SCMDistribution of U blocks of various lengths (12–22 residues) for 1,000 chains of PS1, PS2, and PS3 (degree of polymerization = 100) during digestion by the U XE SCM at the beginning (DE of the native polysaccharide) and at DE values corresponding to the slope change points for the incubations with PEα (SCPEα)3-aThe points of greatest change of slope, SCPEα and SCPEγ, were extracted from the activity versus DE profiles at pH 5.6 in Fig. 1. These curves were separated into two regions that were equated to two straight lines: a plateau region (∂Activity/∂DE = 0) followed by the region with maximum slope (‖∂Activity/∂DE‖ max), and the slope change points correspond to the intersection of these lines. and PEγ (SCPEγ).3-aThe points of greatest change of slope, SCPEα and SCPEγ, were extracted from the activity versus DE profiles at pH 5.6 in Fig. 1. These curves were separated into two regions that were equated to two straight lines: a plateau region (∂Activity/∂DE = 0) followed by the region with maximum slope (‖∂Activity/∂DE‖ max), and the slope change points correspond to the intersection of these lines.3-a The points of greatest change of slope, SCPEα and SCPEγ, were extracted from the activity versus DE profiles at pH 5.6 in Fig. 1. These curves were separated into two regions that were equated to two straight lines: a plateau region (∂Activity/∂DE = 0) followed by the region with maximum slope (‖∂Activity/∂DE‖ max), and the slope change points correspond to the intersection of these lines. Open table in a new tab Table IIIDistribution of U blocks during digestion by MAMDistribution of U blocks of various lengths (12–22 residues) for 1,000 chains of PS1, PS2, and PS3 (degree of polymerization = 100) during digestion by the U XE MAM at the beginning (DE of the native polysaccharide) and at DE values corresponding to the slope change points for the incubations with PEα (SCPEα)2-aThe points of greatest change of slope, SCPEα and SCPEγ, were extracted from the activityversus DE profiles at pH 7.6 in Fig. 1. These curves were separated into two regions that were equated to two straight lines: a plateau region (∂Activity/∂DE = 0) followed by the region with maximum slope (‖∂Activity/∂DE‖ max), and the slope change points correspond to the intersection of these lines. and PEγ (SCPEγ).2-aThe points of greatest change of slope, SCPEα and SCPEγ, were extracted from the activityversus DE profiles at pH 7.6 in Fig. 1. These curves were separated into two regions that were equated to two straight lines: a plateau region (∂Activity/∂DE = 0) followed by the region with maximum slope (‖∂Activity/∂DE‖ max), and the slope change points correspond to the intersection of these lines.2-a The points of greatest change of slope, SCPEα and SCPEγ, were extracted from the activityversus DE profiles at pH 7.6 in Fig. 1. These curves were separated into two regions that were equated to two straight lines: a plateau region (∂Activity/∂DE = 0) followed by the region with maximum slope (‖∂Activity/∂DE‖ max), and the slope change points correspond to the intersection of these lines. Open table in a new tab Distribution of U blocks of various lengths (12–22 residues) for 1,000 chains of PS1, PS2, and PS3 (degree of polymerization = 100) during digestion by the U XE SCM at the beginning (DE of the native polysaccharide) and at DE values corresponding to the slope change points for the incubations with PEα (SCPEα)3-aThe points of greatest change of slope, SCPEα and SCPEγ, were extracted from the activity versus DE profiles at pH 5.6 in Fig. 1. These curves were separated into two regions that were equated to two straight lines: a plateau region (∂Activity/∂DE = 0) followed by the region with maximum slope (‖∂Activity/∂DE‖ max), and the slope change points correspond to the intersection of these lines. and PEγ (SCPEγ).3-aThe points of greatest change of slope, SCPEα and SCPEγ, were extracted from the activity versus DE profiles at pH 5.6 in Fig. 1. These curves were separated into two regions that were equated to two straight lines: a plateau region (∂Activity/∂DE = 0) followed by the region with maximum slope (‖∂Activity/∂DE‖ max), and the slope change points correspond to the intersection of these lines. Distribution of U blocks of various lengths (12–22 residues) for 1,000 chains of PS1, PS2, and PS3 (degree of polymerization = 100) during digestion by the U XE MAM at the beginning (DE of the native polysaccharide) and at DE values corresponding to the slope change points for the incubations with PEα (SCPEα)2-aThe points of greatest change of slope, SCPEα and SCPEγ, were extracted from the activityversus DE profiles at pH 7.6 in Fig. 1. These curves were separated into two regions that were equated to two straight lines: a plateau region (∂Activity/∂DE = 0) followed by the region with maximum slope (‖∂Activity/∂DE‖ max), and the slope change points correspond to the intersection of these lines. and PEγ (SCPEγ).2-aThe points of greatest change of slope, SCPEα and SCPEγ, were extracted from the activityversus DE profiles at pH 7.6 in Fig. 1. These curves were separated into two regions that were equated to two straight lines: a plateau region (∂Activity/∂DE = 0) followed by the region with maximum slope (‖∂Activity/∂DE‖ max), and the slope change points correspond to the intersection of these lines. Assays were performed to identify the factors responsible for the drop in activity at both pHs that were investigated. They consisted of attempts to restore the activity at the end of the deesterification process either by adding new substrate or enzyme molecules in the incubation medium or by changing the pH. Whatever the pH, the addition of new enzyme molecules at the end of the PEα-catalyzed deesterification did not allow resumption of the enzymatic reaction (Fig.2), indicating that the modified substrate generated upon PEα action cannot be further processed. PEα was incubated either at pH 5.6 or at pH 7.6 with PS1 (DE, 74%), and the reaction was allowed to progress until the rate became negligible (Fig.3). PS1 in NaCl was then added to the assay so that the initial conditions were restored. At pH 7.6, the deesterification started again and developed as seen initially. In contrast, at pH 5.6, the reaction started again but progressed much more slowly than it did initially (initial velocity was only a third of that developing at the beginning of the experiment). In this case, the presence in the assay of the enzymatically deesterified substrate,i.e. the presence of acidic blocks, inhibited the deesterification of the native pectin sample. Similar observations were made for PEγ at acidic pH (data not shown) but at pH 7.6, contrary to PEα, the addition of pectin induced no or only a very attenuated resumption of the deesterification process (Fig. 3). PEα was incubated at pH 5.6 with PS1 until the DE was lowered to nearly 60%, and the pH was then adjusted to pH 7.6. The deesterification started again and proceeded rather rapidly until a DE of ∼38% (Fig. 4) was reached. In contrast, if the pH change was made when the reaction rate had become nearly insignificant, which was achieved for a DE around 50%, the deesterification started again but was very short-lived (Fig. 4). However, addition at that time of new pectin molecules induced a strong resumption of the deesterification process (data not shown), similar to the one observed in Fig. 3. At pH 5.6, with both isoforms, addition of PGA in the assay induced a decrease in the velocity. Preliminary experiments carried out with PEα showed that the inhibition was competitive with a Ki estimated to be 4.2 mm galacturonic acid residues. In contrast, at pH 7.6, the two isoforms exhibited a different sensitivity to PGA. With PEα, even high concentrations of PGA (i.e. 7-fold the substrate concentration) did not modify the reaction rate. At this pH, PEγ activity was inhibited by PGA (i.e. 10 mmgalacturonic units induced a 50% decrease of activity). With both isoforms, whatever the pH value, addition of galacturonic acid (the monomer) was without effect. These data confirm the observations made with respect to Fig. 3. At pH 7.6, the presence of new carboxyl groups on the pectin substrate that resulted from enzyme action inhibits the deesterif