Vol. 282, No. 2, November
1, pp. 284&289,1990
Effects of pH on Inactivation of Maize Phosphoenolpyruvate Carboxylase’ Randolph
and M. Kay Black
Received April 12,1990, and in revised form June 26,199O
Maize leaf phosphoenolpyruvate carboxylase (PEPC) is inactivated by incubation at pH’s above neutrality. Both the amount and the rapidity of inactivation increase as the pH rises. The presence of phosphoenolpyruvate (PEP), malate, glucose 6-phosphate and dithiothreitol in the incubation medium give protection to the enzyme. While the presence of PEP during incubation at pH 8 prevents inactivation, the level of PEP in the assay after incubation has no effect on the relative inactivation. When the enzyme is incubated at pH 7 with 5 mM malate (a treatment known to cause dimerization) subsequent assay at saturating levels of MgPEP completely restores activity while assay at 99% inhibition of the same sample, showing that high PEP concentration has reconverted the PEPC to the malate-resistant tetramer. Thus the protective effect of PEP against inactivation at high pH probably is not related to its effect on the aggregation state of the enzyme but rather is due to the presence of PEP at the active site. Protection of PEPC at pH 8 by EDTA and its inactivation by low concentrations of CL? indicates that the loss of activity at high pH probably is in a sense an artifact resulting from the binding to a deprotinated cysteine of heavy metal ions contaminating the enzyme preparation or present in reagents. This suggests that caution should be used in the interpretation of experiments involving PEPC activity at alkaline pH’s. Q ISSO Academic
A variety of experiments have shown that the tetramer of PEPC3 is the form of that enzyme with the high’ This work was supported in part by Grant DCB-8812484 from the National Science Foundation. * To whom correspondence and requests for reprints should be addressed. 3 Abbreviations used: PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; DTT, dithiothreitol; PVP, polyvinylpyrrolidone; glucose 6.phosphate, Glc-6-P; Aces, N-2.acetamido-2284
est activity and that the difference in activity between the tetramer and the dimer may be a major means of regulating its activity (l-7). However, little evidence has been brought forward to show what specific factors may be responsible for controlling the oligomerization of this enzyme so that activity may be regulated. The postulate that regulation occurs because of aggregationldisaggregation of PEPC must also contend with the fact that not all oligomerization changes are reversible and that other regulatory factors have been proposed, including conformational changes in the enzyme resulting from the binding of allosteric ligands (8-11) and phosphorylation of the enzyme (12, 13). It is, of course, possible that if the factors responsible for oligomerization were completely understood, the relationship between oligomerization and other regulatory possibilities would be made clear. The present study is directed toward explication of the reasons underlying the shifts in PEPC activity on dilution at pH values above neutrality. MATERIALS
All chemical and biochemical materials used were purchased Sigma, Boehringer, U. S. Biochemicals, or Aldrich. All materials of the highest purity commercially available.
Enzyme. Highly purified maize PEPC was used for this study. Deribbed maize leaves (200 g fresh weight) were homogenized in 8 ml per gram fresh weight grinding medium consisting of 100 mM Hepes (pH 7.5), 5 mM MgCl,, 5 mM DTT, 1 mM EDTA, 10% glycerol, and 1% (w/v) PVP. Cellular debris was removed by centrifugation. The crude extract was then fractionated with ammonium sulfate. Material precipitating between 30 and 60% ammonium sulfate saturation was dialyzed overnight against 50 mM Hepes (pH 7.5), 1 mM DTT, 10% glycerol, and 5 mM Glc-6-P. The dialysate was passed over a 150.ml bed of Fractogel TSK DEAE 650 M and washed liberally with 50 mM Hepes (pH 7.5), 1 mM DTT, and 50 mM NaCl before eluting PEP carboxylase activity with a wash of buffer containing 250 mM NaCl. Enzyme activity was concentrated by ammonium sulfate precipitation (60% satura-
aminoethanesulfonic acid; Mes, 2.(N-morpholino)ethanesulfonic acid; Tes, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic OAA, oxaloacetate. 000%9861/90
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Effects of pH on Inactivation 1.oo
FIG. 1. Response of maize leaf PEPC to varying pH. Values given are pH’s of incubation mixture consisting of 4 vol of a three buffer mixture (see Materials and Methods) mixed with 1 vol of PEPC in 50 mM Aces, pH 7.0. 0, pH 6.15; A, pH 7.06; +, pH 7.60; 0, pH 7.88; A, pH 8.38; +, pH 9.06.
tion) and 3ml aliquots were desalted/reequilibrated by passing the resuspended pellet through a Bio-Rad acrylagel column (Econo-Pat 10 DG). The partially purified PEP carboxylase, equilibrated with the same buffer used in the batch DEAE procedure, was then applied to a 1.5 X 30-cm DEAE Toyopearl column, washed, and then eluted with a linear 50-200 mM NaCl gradient in the same buffer. The active fractions were pooled, precipitat,ed with ammonium sulfate, and desalted for binding to a 1 X 20.cm hydroxylapatite column (HTP, Bio-Rad) (pH 7.2), 1 mM DTT, and 5% equilibrated with 10 mM K-phosphate glycerol. PEP carboxylase was eluted from the column by incrementing the K-phosphate concentration in steps of 25,50, and 100 mM. The 50 mM K-phosphate wash was concentrated and stored at ~20°C in 25 mM K-phosphate (pH 7.2), 1 mM DTT, 5 mM Glc-6-P, and 10% glycerol. All columns were run at a flow rate of 1 ml/min. Assays. The assay procedure used is that previously reported (14) using both malate dehydrogenase and lactate dehydrogenase as coupling enzymes to minimize the effect of OAA inst,ability. All assays were run at pH 7.0, using 50 mM Aces bufIer and 3.3 mM MgPEP unless otherwise indicated. Incubation. In the studies in which pH was varied from 6 to 9, the buffer used was a combination of Mes, Aces, and Tes, each in 50 mM concentration. For other treat,ments, 50 mM Aces buffer was used at pH 7.0 while 50 mM Tes buffer was used for treatments at pH 8.0. Activity ana/~.si.s. In all cases where significant inactivation occurred, the data were fitted by a nonlinear least squares analysis to the minimal equation of Goldbeter and Koshland (15):
Where FA = fractional residual activity, F, = Fraction of activity susceptible to rapid inactivation, k, = rate constant for rapid inactivation (min-‘), k, = rate constant for slow inactivation and t = time in minutes. Where the total inactivation was less than 100/o, a linear regression line was fitted.
Several reports have noted that inactivation of PEPC by dilution is responsive to pH, with strong effects being noted at pH 8 while relatively little change is seen at pH 7 (16-18). The difference in response to a l/5 dilution over the range from pH 6 to 9 is shown in Fig. 1. Here it is apparent that the loss of activity is both greater and more rapid at alkaline pH’s, diminishing to essentially no effect during a 50-min exposure at pH 6.15. The fitted lines in Fig. 1 use the Koshland inactivation function (15) which provides an estimate of the fraction of activity initially resistant to the treatment (F,) as well as of hl, the rate for the rapid phase of inactivation. The results of the six fitted lines in Fig. 1 show (Table I) that F, diminishes with an increase in pH, so that at pH 9.05 only 12.5% of the original activity remains. More of the activity is destroyed at the higher pH’s and the on rate for the inactivating substance is increased as the pH increases, or the inactivating factor is less effective at lower pH’s. The values in Table I were obtained by fits to duplicate runs, one at low (0.13 mM) MgPEP, the other at saturating (3.3 mM) MgPEP levels. There was no significant difference in the relative rate of inactivation with the two types of assays and in view of the earlier observations of the difference in the recovery of activity by dimerized PEPC at high levels of PEP in the assay (5, 16, 17), this raised questions concerning the nature of the inactivation occurring at high pH’s. Role of PEP in Recovery of PEPC from Inactivation by HighpH The influence of high concentrations of PEP in bringing about increased activity of PEPC has been observed primarily in cases where the enzyme was first induced by preincubation to convert to the dimer or where an
Parameters from Fit of the Data from Fig. 1 to the Koshland Inactivation
6.15 7.06 7.60 7.88 8.38 9.06
1.006 0.902 0.704 0.465 0.175 0.125
0.00015 0.01448 0.2751 0.1603 0.2685 0.7879
(l’i) Correlation coefficient 0.6089 0.6598 0.9672 0.9982 0.9999 0.9999
Note. The value I’, is a measure of the fractional residual activity remaining after the first, rapid phase of inactivation is completed. The k, is the on rate for the rapid phase of inactivation. Lines fitted to duplicate runs, one set assayed at 3.3 mM MgPEP, the other at 0.13 mM MgPEP, both at pH 7.0 with 50 mM Aces.
: 5 5
2 6 0.50 6 2 u 0.25
FIG. 2. Protection of maize PEPC against inactivation at pH 8 by the presence of low (0.13 mM) or high (3.3 mM) PEP. 0, No PEP present; A, low PEP; +, high PEP.
FIG. 3. Effect of level of MgPEP in the assay on maize leaf PEPC which had been incubated for up to 50 min at pH 8 and assayed at pH 7.0. A, 0.13 mM MgPEP; +, 3.3 mM MgPEP.
enzyme already in the dimer form was assayed at high levels of PEP (2,4,5). Since as indicated above the presence of high levels of PEP during assay did not influence the relative inactivation by alkaline pH’s, it seemed desirable to determine whether or not PEPC preincubated at high pH would be protected by the presence of PEP during the preincubation. The results of such an experiment are summarized in Fig. 2, where it may be seen that the presence of a less than saturating level (0.13 mM MgPEP) does give complete protection for short periods and substantial protection against the loss of activity during preincubation at pH 8 for up to 50 min. The higher level of MgPEP (3.3 mM) gives complete protection against loss of activity over the period of the experiment. It is clear from Fig. 2 that PEP in the preincubation mixture is capable of reducing or preventing the inactivation occurring for at least these short times. This raises the question of whether or not the lack of a difference in relative activity at low and high PEP concentrations noted at high pH’s in Fig. 1 applies as well to the standard procedure we have used of preincubation at pH 8 and assay at pH 7. In Fig. 3 it is apparent that when PEPC is preincubated in the same vessel and assayed separately at low PEP concentration and high PEP concentration there is no significant difference between the relative inactivation under the assay conditions, both sets of data being fitted to the same line. Therefore it is apparent that although PEP present during the high pH incubation is capable of protecting PEPC, the presence of PEP in the assay is without effect in inducing recovery
of the enzyme from the high pH inactivation. This unexpected result suggested that the inactivation at high pH was not like the inactivation found when PEPC is exposed to malate (2-5), from which assay high levels of PEP will regenerate the active form of the enzyme (5). Evidence to confirm this was sought directly. As shown in Fig. 1, the inactivation of PEPC is much less at pH 7 than at 8, therefore the enzyme was diluted l/5 with 5 mM malate at pH 7 to induce dimerization and incubated for periods of up to 40 min, being assayed at intervals during the incubation. The results of this experiment are shown in Table II where it is apparent that enzyme assayed at 3.3 mM Mg-
Velocity of PEPC Assayed at Low (0.13 mM) and High (3.3 mM) Levels of MgPEP after Incubation with 5 mM Malate at pH 7 for Varying Times Velocity Time (min) 0 10 15 20 40
11.31 11.30 14.20 15.48 13.81
5.94 0.044 0.029 0.034 0.053
Note. Aliquots removed from the preincubated enzyme solution were transferred at the times indicated to otherwise identical assays containing the two levels of MgPEP.
PEP reaches a velocity as high or higher than the initial rate even after 40 min exposure. However, when enzyme is taken from the same tube and assayed at 0.13 mM MgPEP the activity is found to have decreased more than lOO-fold in 10 min, and it remains at this extremely low level for at least 40 min. The very low level of activity found with a low PEP assay suggests that the dimer induced by dilution with malate is essentially without activity (4). The residual activity found may represent the remaining tetramer present after dilution and equilibration at l/5 in 5 mM malate. In any case these data give strong support for the idea that the dimeric form of PEPC is essentially turned off until it is presented with a high concentration of PEP, when, as shown earlier, it is converted to the tetramer (2-5) and regains its activity. This experiment shows that when PEPC is converted to dimer by malate, it can be reconverted to the active tetramer by high PEP in the assay and suggests that the failure of this reconversion in Fig. 3 means that the inactivation caused by incubation at alkaline pH is not due to formation of the dimer. This tends to confirm our earlier observation (5) that PEPC at high pH is converted rapidly to monomer, and that the monomer then is formed into a “very large” aggregate which is inactive. This also tends to agree with the report of Stiborova and Leblova (18) that the active tetramer of maize PEPC was converted to inactive dimer and monomer at pH 9. From our earlier work (2,5,19) as well as in the results of this study, it is apparent that whatever the nature of the reaction which inactivates PEPC at high pH’s, it cannot be reversed by treatments which influence the dimer/tetramer equilibrium. Although this inactivation appears irreversible, the presence of MgPEP during preincubation is capable of preventing the inactivation in proportion to the concentration present (see Fig. 2) presumably either by fixing the enzyme in an active form, or by blocking a site at which some ligand is binding at high pH to bring about the irreversible inactivation. Malate, Glc-6-phosphate, Inactivation of PEPC
and Cation at pH 8
Two compounds well established as being involved in the oligomerization of PEPC are malate-which inhibits apparently by driving the equilibrium toward dimer (12)-and Glc-6-P, which activates the enzyme by a mechanism which is presently unclear (11). In order to determine whether one of these ligands whose effect on PEPC and its aggregation are well established might interfere with the high pH inactivation, an experiment in which PEPC was diluted l/5 into pH 8 buffer containing 5 mM Mg”+ alone or in combination with 5 mM malate or 5 mM Glc-6-P was performed. The results are shown in Fig. 4, where it is apparent that malate is without effect, not being significantly different from the magne-
FIG. 4. Protection of maize leaf PEPC against inactivation at pH 8 by Glut-6-P and malate. +, Control (no additions during incubation); ~3, 5 mM malate present during incubation; 0, 5 mM Glut-6-P present during incubation.
sium and buffer control, but that Glc-6-P has reduced inactivation by high pH for up to 50 min. The resistance to inactivation conferred by Glc-6-P probably relates to the fact that both MgPEP and MgGlc-6-P are capable of binding at the same site (20, 21) so that the relative protection given by Glc-6-P results from its binding at the normal MgPEP site in agreement with Fig. 2. Another possibility for the high pH inactivation is the deprotonation of a group which could bind divalent cations in an unfavorable position which results in the formation of the large, inactive aggregate (5). To test this possibility PEPC was incubated at pH 8 with 1 mM EDTA and with EDTA plus 5 mM Mg2+ or 5 mM Mn2+. The results are shown in Fig. 5, where it may be seen that the control without EDTA showed the expected inactivation over the 50-min incubation. The presence of EDTA has reduced the inactivation by about half. However, exposure to EDTA plus an excess of either magnesium or managanese has afforded complete protection to the enzyme. This surprising result seems to indicate that if some cation is responsible for the inactivation, which is suggested by the partial protection afforded by 1 mM EDTA (it should be noted that we have observed complete protection from 5 mM EDTA), that neither magnesium nor manganese are the culprits, since they protect the enzyme when EDTA is present, although magnesium, at least, has no effect in the absence of EDTA (see Fig. 4). Role of Thiol
Another example of the ineffectiveness of magnesium in the absence of EDTA is shown in Fig. 6, where the
FIG. 6. Protection of maize leaf PEPC against inactivation at pH 8 by dithiothreitol. f, Control, 5 mM Mg’+; A, 5 mM DTT added to incubation medium.
FIG. 7. Effect of Cu2+ on activity of PEPC at pH ‘7 and pH 8. t, Control, pH 8.0; A, 1 pM CuC12 at pH 8.0; 0, Control at pH 7.0; +, 1 pM CuC12 at pH 7.0.
In this case the reagent was 5 mM dithiothreitol, but the same concentration of mercaptoethanol is equally effective (data not shown). This observation suggests that the high pH inactivation of PEPC, which we think occurs as a result of the formation of a large, inactive and probably unstructured aggregate of the enzyme (5, 19), may involve in some way the formation of S-S bonds starting with monomeric enzyme. Similar conclusions concerning the role of thiols, probably cysteine, in the inactivation of maize PEPC at high pH have been reached by Stiborova and Leblova (18). It seems likely that these results are consistent with an -SH group at the active site and/or the activation site. Heavy Metal Inactivation
control line was incubated with 5 mM Mg”+ and displays the expected inactivation over a 50-min period. The other line in this figure shows that a thiol reagent present in the incubation mixture at pH 8 can completely protect PEPC against the high pH inactivation reaction.
FIG. 5. Protection of maize leaf PEPC against inactivation at pH 8 by EDTA and EDTA plus Mg2+ or Mn’+. +, Control, no additions during incubation; A, 1 mM EDTA added to incubation medium; 0, 1 mM EDTA + 5 mM Mg2+ added to incubation medium; +, 1 mM EDTA + 5 mM Mn2+ added to incubation medium.
Although in view of prior reports (22,23) and the wellestablished toxicity of heavy metals there was little doubt that copper would be highly effective in bringing about the inactivation of PEPC, it seemed desirable to show that this would occur under the conditions used in these studies and also to investigate the relation of Cu2+ toxicity to pH. In Fig. 7 is shown a control line at pH 8, a line showing that 1 PM CuCIZ produces essentially complete inactivation in lo-15 min, a control line at pH 7 with no inactivation found, and a line with 1 yM CuCl, at pH 7 which shows a maximal 25% inactivation during a 50-min exposure. In another experiment (data not shown) the enzyme was incubated with 1 mM EDTA and another incubated with the same concentration of EDTA plus 100 pM ex-
cess CuCl*. The copper caused complete inactivation in 5- to 7-min, showing that while the endogenous inactivation can be relieved with EDTA, the addition of a slight excess of copper ion can cause complete inactivation.
self or, perhaps more likely, that it could be present in the solutions used for preparation and assay of the enzyme as a low level contaminant in commercial chemicals.
The concept of regulation of PEPC activity by aggregation and disaggregation requires that the enzyme be readily equilibrated between, for example, the dimer and the tetramer (2-5) with relatively little loss in the intrinsic activity during cycling between the active tetramer and the low activity or inactive dimer. Such reversibility has been assumed in prior discussions of the possibility of regulation by oligomerization (3, 6). The present study reveals a complication arising from this assumpt,ion. What has been found here might be termed an artifact which probably has no role in the in uiuo regulation of PEPC, but which may be a significant factor in in vitro studies of PEPC activity, particularly as the optimal pH for the maize PEPC is thought to be pH 8.0 (9). The role of thiols in maize PEPC has been demonstrated earlier (18, 22, 23). Briefly stated, the present studies have exposed the fact that at alkaline pH’s an irreversible inactivation of PEPC can be brought about by heavy metals. It appears that this occurs when a cysteine in the active site is deprotonated, making it receptive to reaction with a heavy metal ion, or at least with CL?+. While there is certainly copper present in the living cell, it seems likely that the pH seldom reaches a level high enough to cause substantial inactivation, and in any case protective ligands such as PEP, Glc-6-P, and innocuous cations probably abound in the cell. However, in an assay, it is clear that even a relatively small amount of copper can, under the right circumstances, produce a very significant decrease in the activity which would otherwise be found and could lead to erroneous conclusions concerning the effect of the treatment being evaluated. This possibility is particularly strong when the assay is initiated by the addition of substrate so that the enzyme is exposed to endogenous heavy metals without the protection afforded by the presence of PEP. The source of the copper is an open question, but the relatively small amount required (18) probably indicates that it could be provided by the enzyme preparation it-
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