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Properties and reaction mechanism of C4 leaf pyruvate,Pi dikinase
OF BIOCHEMISTRY AND BIOPHYSICS Vol. 239, No. 1, May 15, pp. 53-62, 1985
ARCHIVES
Properties and Reaction Mechanism of C4 Leaf Pyruvate,Pi Dikinase C. L. D. JENKINS Division
of Plant
Industry,
AND M. D. HATCH’
CSIRO, GPO Box 1600, Canberra, A.C. T 2601, Australia
Received December 17, 1984
The properties and reaction mechanism of maize leaf pyruvate,Pi clikinase are described. K,,, values were determined for the forward reaction substrates, pyruvate, ATP, and Pi, at pH 7.4 and 8.0 and for reverse reaction substrates at pH 7.4. Enzyme activity was almost totally dependent on added monovalent cations in both directions. NH: was most effective, with K, values of about 0.38 mM for the forward reaction and 2 mM for the reverse reaction. K+ also completely activated the enzyme in the forward direction (K, = 8 mM) but only partially activated in the reverse direction. Na+ had little effect on either reaction. The pH optimum for the forward reaction was about 8.2; the reverse reaction optimum was about 6.9. Maximum activity for the reverse direction was about twice the maximum forward direction rate. From data on the requirements for the ATP-AMP exchange reaction, on the mechanism of inhibition of the forward reaction by PEP, AMP, and PPi, and from the kinetics of the interaction of varying certain substrate pairs, it was concluded that the maize leaf pyruvate,Pi clikinase reaction proceeded by the two-step Bi Bi Uni Uni mechanism. o 19% Academic This differs from the mechanism of catalysis by the bacterial enzyme. Press, Inc.
Pyruvate,Pi dikinase (EC.2.7.9.1) catalyses the conversion of pyruvate to phosphoenolpyruvate (PEP)’ as follows: Pyruvate
from maize (1) or sugarcane (5) and essentially pure enzyme from maize (6). Studies of the partial reactions catalyzed by pyruvate,Pi dikinase by following racliotracer exchange supported the view that catalysis by the sugarcane leaf enzyme involves a sequential mechanism in which ATP and Pi react to give an enzymeP (E-P) intermediate (5). In the second partial reaction this intermediate apparently reacts with pyruvate to give PEP by a so-called “ping-pang” reaction, thus ATP+Pi+E 5 E-P + AMP + PPi
+ ATP + Pi =PEP
+ AMP + PPi
The enzyme was first demonstrated in the leaves of C4 plants (l), where it operates in mesophyll cell chloroplasts to catalyze the light-dependent conversion of pyruvate to PEP [see Ref. (2)]. It also occurs in various bacteria [see Ref. (3)] and has more recently been shown to occur in some other plant tissues [see Ref. (4)]. Various physical and kinetic properties of C4 leaf pyruvate,Pi clikinase have been examined using partially purified enzyme
E-P + pyruvate
= E + PEP.
Surprisingly, catalysis by bacterial pyruvate,Pi clikinase proceeds by a different mechanism designated as Tri Uni Uni Ping Pong (7). This sequence involves a reaction with ATP to give E-PP plus AMP followed by a reaction of E-PP with Pi to give E-P plus PPi, followed by a reaction of E-P with pyruvate as described above.
’ To whom correspondence should be addressed. ’ Abbreviations used: PEP, phosphoenolpyruvate; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. 53
0003-9861/85 $3.00 Copyright All rights
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
54
JENKINS
AND
We have recently described a complex mechanism for reversibly inactivating maize leaf pyruvate,Pi dikinase which accounts for the light-dark regulation of this enzyme in leaves (8, 9). This involves inactivation by ADP-dependent phosphorylation of an enzyme threonine residue and reactivation by phosphorolytic cleavage of this ester bond. These reactions depend on the existing phosphorylation status of pyruvate,Pi dikinase at its catalytic site and are controlled in various ways by substrates or products of this reaction. In view of these findings we sought more details about some of the properties of the maize leaf enzyme. We also used additional procedures to confirm that the maize pyruvate,Pi dikinase has a similar mechanism of catalysis to the one described earlier (see above) for the enzyme from sugarcane. MATERIALS
AND
METHODS
Plant tissue and materials.Maize plants (Zeo mays, variety Dekalb XL 81) were grown in soil in a greenhouse maintained between 20 and 30°C. Biochemicals and reagent enzymes were obtained from Boehringer-Mannheim, Australia or Sigma Chemical Company. PEP carboxylase used for the assay of pyruvate,Pi dikinase was prepared essentially as previously described (lo), except that the active fractions from DEAE-Sepharose-6B were concentrated and then chromatographed on a 180-ml column of Sephadex G-200 to obtain fractions free of pyrophosphatase. Pur$cntion of pyruvate,Pi dikinase. Pyruvate,Pi dikinase was extracted from maize leaves and then fractionated by precipitation with (NH&SO4 and Sephacryl S-300 column chromatography as previously described (ll), except that 5 mM dithiothreitol was used instead of 50 mM 2-mercaptoethanol and solutions were maintained anaerobic with nitrogen. The most active fractions from Sephacryl S-300 were pooled and then chromatographed on a DEAESepharose-6B column essentially as described by Burnell and Hatch (12). Peak fractions were at least 70% pure as judged by electrophoresis on dissociating acrylamide gels [see Ref. (8)]. Fractions free of pyrophosphatase were obtained by this procedure although the complete removal of this enzyme was not achieved in all preparations. Active fractions were stored at 2°C as a suspension in 65% saturated (NH,)zSOI solution. Alternatively, protein concentrated by precipitation with (NH&SO4 and then
HATCH
treated on a Sephadex G-25 column to remove (NH&SO, was stored at -20°C after adding an equal volume of glycerol. Assay of pllrmvate,Pi dikinase. All assays were conducted at 25°C. For the forward direction (pyruvate to PEP) the routine method was based on the spectrophotometric measurement of NADH oxidation associated with conversion of PEP to oxaloacetate (via PEP carboxylase) followed by reduction to malate via NAD-malate dehydrogenase (5). Reactions (1 ml) contained 25 mM Hepes-KOH, pH 8.0 (or pH 7.4 where specified), 8 mM MgSO,, 10 mM dithiothreitol, 10 mM NaHCOa, 1 mM glucose-6-P, 5 mM (NH1)zSOl, 2 mM pyruvate, 1 mM ATP, 2.5 mM potassium phosphate, 0.2 mM NADH, and about 0.25 unit of PEP carboxylase plus 1 unit of NAD-malate dehydrogenase. Generally, reactions were started by adding pyruvate,Pi dikinase. Where PEP inhibition of the forward reaction was examined activity was measured as AMP produced in reactions of a composition described above except that pyruvate was 0.5 mM, ATP was 0.4 mM, and NaHCOa, glucose-6-P, NADH, and the coupling enzymes were omitted. These reactions were incubated with varying concentrations of PEP and then stopped by adding HCl to give a final concentration of 0.025 M, and then stored at 0°C if they were to be immediately processed or frozen at -20°C. Samples were neutralized with KOH and then AMP was measured by following the total change of absorbance at 340 nm in reactions containing ATP, PEP, adenylate kinase, pyruvate kinase, NADH, and lactate dehydrogenase. These reaction mixtures for measuring AMP were essentially the same as described earlier for the assay of adenylate kinase (13). Pyruvate and any traces of ADP present in the samples were first consumed by mixing the sample with the reaction mixture from which adenylate kinase had been omitted. The absorbance change following the addition of adenylate kinase was then determined. For the reverse reaction (PEP to pyruvate) the assay was based on the previously described procedure (5) in which pyruvate production was measured spectrophotometrically by the NADH oxidized during conversion of pyruvate to lactate via lactate dehydrogenase. Reactions contained 25 mM Hepes-KOH, pH 7.4 (or 25 mM Tris-HCl, pH 8.0 were specified), 6 mM MgSO,, 10 mM dithiothreitol, 25 mM NH&l, 1 mM PEP, 0.5 mM AMP, 1 mM PPi, and 2 units of lactate dehydrogenase. Reactions were usually started by the addition of pyruvate,Pi dikinase. For ‘some experiments the rate of absorbance change at 340 nm observed with all reactants except AMP was subtracted from the rate observed with the complete reaction. Since pyruvate,Pi dikinase had a very high affinity for AMP the experiments conducted to determine
C4 LEAF
PYRUVATEJ’,
its K,, and others designed to determine the kinetics of interaction between AMP and PPi, employed an assay procedure which measured the conversion of [‘%]AMP to ATP. Reactions were as described above except that the PPi concentration was 0.5 mM and the [‘%]AMP concentration was varied. Reactions of 0.2 ml volume were started by adding 20 pl of pyruvate,Pi dikinase, previously diluted to be equivalent to an activity of about 0.0003 absorbance unit change min-’ for 20 pl of enzyme in a l-ml reaction measured by the spectrophotometric assay at 340 nm. Reactions were stopped at intervals by adding 20 pl of 2 M trifluoroacetic acid and stored on ice or at -80°C. Notably this acid is more volatile than water so that acidity does not increase when samples were subsequently spotted on thin-layer chromatogram plates. Samples of these reactions were spotted on polyethyleneimine-cellulose thin-layer plates, dried, and then developed in a solvent containing 1 M LiCl and 0.5 M acetic acid. Prior to spotting the samples on chromatograms the acidified reactions were supplemented with additional unlabeled AMP and ATP to facilitate subsequent detection of these compounds by viewing the developed chromatograms under ultraviolet light. The areas containing ATP and AMP were cut out and counted by the liquid scintillation procedure. Rates were calculated from the percentage conversion of [%]AMP to ATP. Reactions con[“CjAMP-ATP exchange react&n. tained 40 mM Hepes-KOH of varying pH, 7 mM dithiothreitol, 7 mM MgCl*, 5 mM NH&l, 0.66 mM ATP, and 0.16 mM [r4C]AMP, in a total volume of 0.3 ml. Orthophosphate, PPi, and PEP were added at the concentrations specified in Table III. Reactions were started by the addition of enzyme and then incubated at 25°C for intervals before stopping by the addition of 20 pl of 2 M trifluoroacetic acid and stored at -20°C. Rates of exchange were measured by determining the label appearing in ATP by chromatography as described above. Determining monovalent metal ion requirements. For studying the response to monovalent inorganic cations the following procedure was adopted to free reactions of cations present in reagents. Forward and reverse direction assay mixtures were made up except that enzymes, buffer, NADH, NaHC03, NH4Cl, MgS04, and, in the case of the reverse assay, AMP were omitted. The mixtures were passed through a 4-ml column of Dowex-50 (H+ form) to remove all cations and then 2 M Tris was added to adjust the mixtures to the desired pH (7.4 or 8.0). For the forward direction assay the following were then added to complete a reaction of 1 ml: 25 ~1 of 0.2 M MgS04, 20 al of 10 mM barium NADH, and 50 (~1of 0.1 M triethylamine bicarbonate, and then PEP carboxylase-malate dehydrogenase and also pyruvate,Pi dikinase previously treated on a Sephadex G-25
55
DIKINASE
column equilibrated with 10 mM Tris-HCl, pH 7.5, containing 5 mM MgSOl and 5 mM dithiothreitol. This treatment was designed to remove NH: and other monovalent metal ions from the enzyme preparations. A similar procedure was adopted for the reverse direction assay except that 25 ~1 of 0.2~ MgS04, 10 ~1 of lactate dehydrogenase (Sephadex G-25 treated), and 20 ~1 of 10 mM barium NADH were added before the pyruvate,Pi dikinase.
RESULTS
AND
DISCUSSION
Substrate Afinities and Product Inhibition Apparent Michaelis constants were determined for pyruvate, ATP, and Pi at pH 8.1 and 7.4, and for PEP, AMP, and PPi at 7.4 (Fig. 1 and Table I). The Lineweaver-Burk double-reciprocal plots shown in Fig. 1 for the forward direction substrates, pyruvate, ATP, and Pi, are for pH 8.1 only. All plots were linear. The values obtained for the forward reaction substrates differ substantially from some of those reported in earlier studies [90 PM for ATP at pH 8.3 (l), 250 PM for pyruvate, 15 PM for ATP, and 1.5 mM for Pi at pH 7.5 (S)]. Reverse reaction K, values for PEP and PPi were in close agreement with those reported earlier for the enzyme from sugarcane (15) and maize (6). The Km for AMP is very low and was not determined previously. We obtained values of 1.1 PM at pH 8.0 and 0.85 PM at pH 7.4. This may mean that the enzyme always exists as the AMP complex in vivo. Inhibition of the forward reaction by all three products, PEP, AMP, and PPi, briefly reported earlier (5), was examined in more detail. The data showing the interactions of PEP with pyruvate, of AMP with ATP, and of PPi with Pi, are shown in the form of Linewaver-Burk reciprocal plots in a later section of this paper (see Fig. 4). The results for Ki values and types of inhibition are summarized in Table I. For PEP the Ki was 150 PM and inhibition was linearly competitive with respect to pyruvate. The Ki was 130 PM for AMP and 320 PM for PPip and inhibition was noncompetitive with respect to ATP and Pi, respectively.
56
JENKINS
h:
6
,z E c 0 ”
4
5
2
0
0
6
12
l/Pyruvate
l/PEP
18
0
(mM”)
(mM-‘)
AND
8
HATCH
16 1 /ATP
(mM-‘)
l/AMP
(PM-‘)
24
I
I
I
1
I
1
0
1
2
3
4
5
~/PI (mM-‘J
FIG. 1. Lineweaver-Burk plots for the substrates of pyruvate& dikinase in the forward and reverse directions. For the forward direction (substrates: pyruvate, ATP, and Pi) the pH was 8.0 and for the reverse direction the pH was 7.4. Other details of assay procedures and reaction mixture composition are provided under Materials and Methods.
TABLE
Requirement for Monovalent Cations
I
Km VALUESFOR SUBSTRATESOF PYRUVATE, Pi DIKINASEANDTHE Ki VALUESFOR PRODUCT INHIBITORS Km (~4 Substrate
pH 8.1
Forward reaction Pyruvate ATP pi
82 32 380
Reverse reaction PEP AMP PPi
-
1.1
Inhibitor
K (/.M
Forward reaction PEPa AMP PPi
150 130 320
pH 7.4
92 48 560 160 0.85 46
(C versus pyruvate)* (NC versus ATP) (NC versus Pi)
EPEP inhibition was mixed (noncompetitive) with respect to both Pi and ATP. “C and NC refer to competitive and noncompetitive inhibition, respectively.
Maize leaf pyrllVRk?~i dikinase requires the divalent metal ion Mg2+ for activity (1); at least for reverse direction activity a monovalent cation is also essential although only marginal stimulation of the forward reaction was reported (6). We further investigated these requirements. Using assay mixtures treated to remove metal ions associated with reagents we showed that both the forward and reverse directions of the pyruvate,Pi dikinase reaction are almost totally dependent on added monovalent cations. For the forward direction reaction assayed at pH 8.1 this requirement was satisfied by relatively low concentrations of NH:, or much higher K+; Na+ was much less effective (Fig. 2). For the reverse direction much higher NH: was required for maximum activity; there was a partial response to K+ but no effect of Na+. Table II summarizes the responses to NH:, K+ and Na+ seen in Fig. 2 and also includes data for the forward reaction at pH 7.4; also recorded are K, values where responses
Cd LEAF
PYRUVATE,Pi
Catlo”,rnM,
cmon,rnM,
FIG. 2. Monovalent cation requirements for pyruvate,Pi dikinase activity in the forward (pyruvate to PEP) and reverse directions. Reactions were treated to remove monovalent metal ions as described under Materials and Methods. All cations were provided as their chloride salts.
were significant. The K, values for K+ in the forward direction (determined from double-reciprocal plots) were about 30 times those for NH: (Table II). Notably, the K, value for NH: for the reverse reaction was about 10 times that for the forward reaction. In other experiments we confirmed an earlier observation (1) that the divalent metal ion requirement for the forward reaction was satisfied by a Mg2+ concentration of about 3 mM in excess of the TABLE
DIKINASE
57
ATP concentration. Additional Mg2+ up to 50 mM had no effect in reducing the requirement for NH: or other monovalent metal ions. These data on monovalent metal ion requirements are similar to those obtained for bacterial pyruvate,Pi dikinase (14). However, our results differ quantitatively from those reported by Sugiyama (6) for metal ion responses of the reverse reaction catalyzed by maize leaf enzyme. He recorded a K, of about 5 mM for NH: at pH 7.4 and obtained much higher degrees of activation with K+ and also Na+. Although NH: is the most effective activator of pyruvate,Pi dikinase in vitro it may not contribute to the activation of this enzyme in vivo. We found that the K, for NH: was about 0.3 mM for the forward reaction and much higher for the reverse reaction. However, estimates of metabolic compartment NH: concentrations in vivo, based on NH3 exchange with leaf tissue, would indicate concentrations of only about 30 j&M NH: at pH 7.0 (15). At diffusion equilibrium this concentration would be even less at pH 8, about the presumed pH of the chloroplast stroma in the light (16). However, at least the forward reaction for pyruvate,Pi dikinase is equally effectively activated by II
MONOVALENT METAL ION REQUIREMENT FOR PYRUVATE, Pi DIKINASE Activation
Cation and pH Forward reaction NH:, pH 8.1 KC, pH 8.1 Na+, pH 8.1 NH:, pH 7.4 K+, pH 7.4 Na+, pH 7.4 Reverse reactionb NH;, pH 7.4 K+, pH 7.4 Na+, pH 7.4
(Pyruvate
response
Ka Cm@
to PEP) >20-fold >20-fold ~25% activated by 40 InM” >20-fold >20-fold About 15% activated by 40 >20-fold About 40% activated No effect
DPercentage activation refers to the activity relative to the maximum Concentrations above about 80 mM K+ or Na+ inhibit activity. bAt pH 8.1 the reverse reaction is activated only about seven-fold essentially no effect.
by 40
0.38 10 0.25 8 -
mM”
2.0 10 -
mMa
observed with optimal by NH:
while
NH;
or KC.
K+ and Na+ have
58
JENKINS
K+ as by NH: although concentrations of up to 80 mM K+ are required for full activity. This presumably presents no problem in viva since the natural K+ concentration in chloroplasts is estimated to be about 100 InM (17).
Efect of Varying pH Although leaf pyruvate,Pi dikinase has been studied in some detail (1, 5, 6) little information has been reported about the effect of varying pH on the forward direction and none is available for the reverse direction. As previously reported (1) the optimum for the forward reaction was about 8.2; there was very little activity below pH 7 (Fig. 3). Reverse direction activity was optimal at about pH 6.8 and activity declined on the alkaline side of this pH so that there was only about 10% of maximum activity at pH 8.4. Activities for the forward and reverse reactions were about equal at pH 7.6. The marked opposite effects on the forward and reverse reactions of increasing pH in the range from about 6.8 to 8.3 are probably related to the fact that the forward reaction results in a stoichiometric production of 2H+ for each pyruvate converted to PEP (18). In C4 leaves pyru-
AND
HATCH
vate,Pi dikinase operates in mesophyll chloroplasts in the direction of PEP synthesis (2). Notably, activity will be optimal for this direction at the predicted pH of about 8.2 for the stroma in the light (see above), while the reverse reaction activity will be relatively low. It may also be significant that the likely NH: levels in chloroplasts will be far too low to activate pyruvate,Pi dikinase in this direction (see above discussion). Since K+ only partially activates the reverse reaction and Na+ not all, the combination of this limited activation and the high stromal pH may render activity in the direction of pyruvate synthesis insignificant in vivo.
Reaction Mechanism The following experiments were conducted to test the validity of an earlier conclusion (5) that the leaf pyruvate,Pi dikinase proceeds by a two-step mechanism (see Introduction) rather than the three-step mechanism reported for the bacterial enzyme (7). For the leaf enzyme the mechanism proposed [Bi Bi Uni Uni according to the nomenclature of Cleland, Ref. (19)] is E + ATP + Pi * E-P + AMP + PPi E-P + pyruvate
+ E + PEP.
[Al
The mechanism proposed for the bacterial enzyme (Tri Uni Uni) is E + ATP = E-PP + AMP E-PP + Pi C= E-P + PPi E-P + pyruvate
7.0
7.5
80
85
PH
FIG. 3. Effect of varying pH on the activity of pyruvate,Pi dikinase in the forward (pyruvate to PEP) and reverse directions. All reactions were buffered with a 20 mM Hepes-20 mhl Tricine mixture adjusted with KOH to the desired pH. The actual pH of reactions were measured after assays were completed.
[Bl
c E + PEP.
A critical reaction for distinguishing between these mechanisms is the partial reaction leading to [14C]AMP-ATP exchange. Predictions are that Pi and PPi will be required for the AMP-ATP exchange if mechanism A is operative [see the above scheme and Refs. (5, 7)]. With mechanism B there will be no other requirements but Pi or PEP will inhibit due to conversion of the enzyme to the E-P form, which does not participate in this particular exchange reaction. We con-
Cd LEAF
PYRUVATE,P,
firmed our earlier observation (5) that with the leaf pyruvate,Pi dikinase this [14C]AMP-ATP exchange is stimulated several fold by the combined addition of Pi and PPi (Table III). Increases of UP to sixfold were observed although the response varied between experiments and tended to be less at lower pH. Although reagents were treated to remove contaminating Pi and PPi (see footnote, Table III), traces of these compounds may have contributed to some of the activity seen in the control reactions. However, the fact that there was usually little response to adding either Pi or PPi separately supported the view that there was little contamination by Pi and PPi in the control reactions (Table III). In this respect the exception was the substantial increase seen by adding Pi alone at pH 7.0. A similar response to Pi was seen in a separate experiment not quoted here, but we have no simple explanation for this TABLE
III
REQUIREMENT FOR CATALYSIS EXCHANGE
REACTION
OF [%]AMP-ATP BY PYRUVATE, Pi DIKINASE
Exchange activity (rmol ml-’ 4 min-‘) Expt. 1
Expt. 2
Additions to basic reaction”
PD 8.0
PD 7.5
PH 8.2
PD 7.0
None pi PPi Pi + PPi Pi + PPi + PEPb PEP
0.77 0.91 0.77 2.9 3.5 0.77
0.70 0.91 0.73 2.0 5.2 0.81
0.38 0.38 0.35 2.4 3.2 0.38
0.38 1.2 0.33 2.3 9.1 0.42
“Pi concentration was 1 mM for Expt. 1 and 2.5 mM for Expt. 2. Concentrations of PPi and PEP were 0.08 and 1 mM, respectively. For these experiments the ATP used was treated on a Sephadex G10 column to remove Pi and PPi and the Pi stock solution was chromatographed on DEAE-Sephadex (8) to remove PPi. b This reaction contains all reverse direction substrates (PEP, AMP, and PPJ and hence measures reverse reaction activity.
DIKINASE
59
result. Notably, the rate of the [14C]AMPATP exchange was close to the rate of the reverse reaction activity (given by reactions supplemented with PPi plus PEP) at about pH 8.0 but was much less than the reverse reaction rate at lower pH. Presumably this is due to increasing reverse reaction activity as the pH is reduced (see above and Fig. 3). This increase may not be accompanied by increased exchange reaction activity due to the constraint imposed by the forward reaction. The origin of the AMP-ATP exchange activity seen in the control reactions containing no added Pi or PPi (see Table III) is uncertain. As discussed above, if this was due solely to contaminating Pi and PPi then larger responses to adding Pi or PPi alone might have been expected. On the other hand, if this activity was due to the first partial reaction of mechanism B then Pi and PEP would be expected to inhibit (see above) and no such inhibition was observed (Table III). As discussed in detail by Milner and Wood (7) the alternative reaction mechanism for pyruvate,Pi dikinase may also be distinguished by the kinetics of inhibition of the forward reaction by the products, PEP, AMP, and PPi, and by the kinetic patterns resulting from varying certain substrate pairs. We found that with the leaf pyruvate,Pi dikinase PEP inhibited competitively with respect to pyruvate and that AMP and PPi inhibited noncompetitively with respect to ATP and Pi, respectively (Fig. 4). The results for AMP and PPi inhibition are in accordance with the predictions outlined by Milner and Wood (7) for mechanism A. They are not consistent with the operation of mechanism B, where competitive inhibition is predicted. Different patterns of inhibition of the forward reaction by PEP have been predicted depending on whether the partial reactions of mechanism A, and also of mechanism B, occur at a common overlapping site or at functionally independent sites [see Ref. (7)]. Our observation that PEP inhibits competitively with respect
60
JENKINS
OmM
o0
AND
HATCH
PEP
-1
2 1 /Pyruvate
3
4 (mM-‘)
5
0
4
8 1 /ATP
12
160
1
2
3
l/Pi
(mM”)
4
5
(mM”)
FIG. 4. Kinetics of the inhibition of the forward reaction of pyruvate,Pi dikinase by the products, PEP, AMP, and PPi. The pH was 8.0 and other details of assay procedures are described under Materials and Methods.
to pyruvate (Fig. 4) indicates that the second partial reaction of mechanism A (pyruvate-PEP exchange) takes place at a site which is functionally independent of the one catalyzing the first partial reaction. Further support for this alternative was provided by the observation that PEP inhibited noncompetitively with respect to ATP and Pi (results not shown). As discussed by Milner and Wood (7), there are only two substrate pairs which interact to give kinetics that distinguish mechanism A from mechanism B. These are AMP-PPi and ATP-Pi. For the former pair the Lineweaver-Burk reciprocal plot we obtained when both AMP and PPi were varied gave intersecting lines (Fig. 5). This agrees with the operation of mechanism A; mechanism B should give parallel lines. The prediction for the plots obtained when ATP and Pi are simultaneously varied are for intersecting lines with mechanism A and parallel lines with mechanism B. The data we obtained in several experiments consistently gave plots showing essentially parallel lines (Fig. 5), a result apparently not consistent with the operation of mechanism A. This pattern indicates that ATP and Pi interact to affect the binding of each other to the enzyme, with affinity increasing as the concentration of the other substrate decreases (see Fig. 5). This may be explained if mechanism A operates but the binding of ATP and Pi to free enzyme is higher
than the binding to the Es Pi and E * ATP forms, respectively. CONCLUDING
COMMENTS
Maize leaf pyruvate,Pi dikinase operates in the light in chloroplasts to convert pyruvate to PEP. For spinach chloroplasts at least, the pH in the stroma varies between about pH 7.2 in the dark and about pH 8.2 in the light (16). Clearly, the pH response for the forward reaction of pyruvate,Pi dikinase (see Fig. 3) ideally suits this enzyme for this particular role. As noted above, the decreasing reverse reaction activity as pH increases, and the likelihood of only partial activation of the
“0
10
20
IlnTPlmM’)
30
0
1
2 ,/AMP
3
4
#o.v’I
FIG. 5. Kinetic responses to simultaneously varying the substrate pairs ATP and Pi for the forward reaction and AMP and PPi for the reverse reaction assessed by Lineweaver-Burk reciprocal plots. The pH was 8.0 and other details are provided under Materials and Methods.
Cd LEAF PYRUVATEQi
reverse reaction by monovalent metal ions in viva (see above) would combine to make activity in this direction negligible in the light. Other evidence would suggest that these particular pH-activity responses were probably not evolved especially to optimize the operation of this particular enzyme in this particular situation. For instance, pyruvate,Pi dikinase from bacteria, including the enzyme from Bacteroides symbioses which apparently operates to convert PEP to pyruvate in viva, have similar pH response characteristics. Like the maize leaf pyruvate,Pi dikinase these bacterial enzymes are also more active in the reverse direction than the forward direction when each activity is determined under optimal conditions. As already discussed, it appears most likely that the pH responses are largely a consequence of the involvement of H+ in the reaction. There appears to be no obvious significance in the differences in K, for the substrates of pyruvate,Pi dikinase from different sources. There were substantial differences in some of the K, values for pyruvate, ATP, and Pi with enzyme from such diverse sources as maize leaf, wheat seed (2), and various bacteria [see Ref. (3)], all of which operate in viva for PEP synthesis. Notably, the K, values for substrates in the B. symbiosis enzyme [believed to operate in the reverse direction in viva; see Ref. (21)] fell within the range of values recorded for the other enzymes. The K, for AMP for all these enzymes is very low (range, 1-15 PM). The only explanation we can offer for this is that strong binding of AMP may increase catalytic efficiency through the effect described by Albery and Knowles (22) of reducing the free energy difference between kinetically critical intermediate forms of the overall reaction. Earlier studies (5) of exchange reactions catalyzed by the sugar-cane leaf pyruvate,Pi dikinase indicated a Bi Bi Uni Uni mechanism (referred to above as mechanism A). Later evidence [see Ref. (7)] indicated that bacterial pyruvate,Pi dikinase operated by a Tri Uni Uni mechanism
61
DIKINASE
(mechanism B described above). Our present studies confirmed by various additional procedures the operation of the A-type mechanism for the maize leaf enzyme. The significance of this difference remains to be resolved. One possibility is that the A-type mechanism may be important for the processes leading to the light-dark regulation of pyruvate,Pi dikinase. For this complex process only pyruvategidikinase phosphorylated at the catalytic-site histidine is inactivated by ADP-mediated phosphorylation at a separate site on the enzyme; by contrast, Pimediated activation by phosphorolytic dephosphorylation is most rapid with the noncatalytically phosphorylated form of the enzyme (8, 9, 23). Notably, inactive pyruvate,Pi dikinase does not catalyze the pyruvate-PEP exchange reaction and hence cannot be dephosphorylated at its catalytic site by pyruvate (23); however, this inactive enzyme does catalyze the first exchange reaction between ATP + Pi and AMP + PPi (see mechanism A above). Consequently, AMP + PPi can react with the inactive enzyme to remove the catalytic-site phosphate group and hence convert it to a form that is more rapidly activated by a reaction with Pi. ACKNOWLEDGMENT We acknowledge the expert technical assistance of Tony Agostino. REFERENCES 1. HATCH, M. D., AND SLACK, C. R. (1968) &o&em J. 106, 141-146. 2. HATCH, M. D., AND OSMOND,C. B. (1976) in Encyclopedia of Plant Physiology (New Series) (Stocking, C. R., and Heber, U., eds.), Vol. 3, pp. 144-184, Springer-Verlag, Heidelberg. 3. WOOD,W. A. (editor) (1975) in Methods in Enzymology (Wood, W. A., ed.), Vol. 42, pp. 18’7212, Academic Press, New York. 4. EDWARDS,G. E., NAKAMOTO,H., BURNELL,J. N., AND HATCH, M. D. (1985) Annu. Rev. Plant Physiol, in press. 5. ANDREWS, T. J., AND HATCH, M. D. (1969) Biochem. J. 114, 117-125. 6. SUGIYAMA,T. (1973) Biochedst?y 12,2862-2868. 7. MILNER, Y., AND WOOD, H. G. (1976) J. Bid Chem 251, 7920-7928.
62
JENKINS
8. ASHTON, A. R., BURNELL, J. N., AND HATCH, M. D. (1984) Arch. Biochem. Biophys. 230,492503. 9. BURNELL, J. N., AND HATCH, M. D. (1984) Arch Biochem. Biophys. 231,1’75-182. 10. HATCH, M. D., AND HELDT, H. W. (1985) AnaL Biockem., in press. 11. HATCH, M. D. (1979) Aust. J. Plant Physid 6, 60’7-619. 12. BURNELL, J. N., AND HATCH, M. D. (1985) Arch Biochem. Biqphys., in press. 13. HATCH, M. D. (1982) Aust. .I Plant Physid 9, 287-296. 14. MICHAELS, G., MILNER, Y., MOSKOVITZ, B. R., AND WOOD, H. G. (1978) J. BioL Chem. 253, 76567661. 15. FARQUHAR, G. D., FIRTH, P. M., WETSELAAR, R., AND WIER, B. (1980) Plant PhysioL 66, 710714.
AND
HATCH
16. HELLIT, H. W., WERDEN, K., MILOVANCEV, M., AND GELLER, G. (1973) Biochem Biophys. Ada 314,224-241. 17. DEMMIG, B., AND GIMMLER, H. (1983) Plant Physiol. 73,169-174. 18. REEVES, R. (1968) J. Bid
Chem 243, 3202-3204.
19. CLELAND, W. W. (1970) in The Enzymes (Boyer, P. D., ed.), Vol. 2, pp. l-65, Academic Press, New York. 20. MEYER, A. O., KELLY, G. J., AND LATZKO, E. (1982) Plant PhysioL 69, 7-10. 21. WOOD, H. G., O’BRIEN, W. E., AND MICHAELS, G. (1977) A&J. EnzymoL 45, 85-155. 22. ALBERY, W. J., AND KNOWLES, J. R. (1976) Biochemistry 15,5631-5640. 23. BURNELL, J. N. (1984) B&hem. Commun 120. 559-565.
Biophys.
Res.
ARCHIVES
Properties and Reaction Mechanism of C4 Leaf Pyruvate,Pi Dikinase C. L. D. JENKINS Division
of Plant
Industry,
AND M. D. HATCH’
CSIRO, GPO Box 1600, Canberra, A.C. T 2601, Australia
Received December 17, 1984
The properties and reaction mechanism of maize leaf pyruvate,Pi clikinase are described. K,,, values were determined for the forward reaction substrates, pyruvate, ATP, and Pi, at pH 7.4 and 8.0 and for reverse reaction substrates at pH 7.4. Enzyme activity was almost totally dependent on added monovalent cations in both directions. NH: was most effective, with K, values of about 0.38 mM for the forward reaction and 2 mM for the reverse reaction. K+ also completely activated the enzyme in the forward direction (K, = 8 mM) but only partially activated in the reverse direction. Na+ had little effect on either reaction. The pH optimum for the forward reaction was about 8.2; the reverse reaction optimum was about 6.9. Maximum activity for the reverse direction was about twice the maximum forward direction rate. From data on the requirements for the ATP-AMP exchange reaction, on the mechanism of inhibition of the forward reaction by PEP, AMP, and PPi, and from the kinetics of the interaction of varying certain substrate pairs, it was concluded that the maize leaf pyruvate,Pi clikinase reaction proceeded by the two-step Bi Bi Uni Uni mechanism. o 19% Academic This differs from the mechanism of catalysis by the bacterial enzyme. Press, Inc.
Pyruvate,Pi dikinase (EC.2.7.9.1) catalyses the conversion of pyruvate to phosphoenolpyruvate (PEP)’ as follows: Pyruvate
from maize (1) or sugarcane (5) and essentially pure enzyme from maize (6). Studies of the partial reactions catalyzed by pyruvate,Pi dikinase by following racliotracer exchange supported the view that catalysis by the sugarcane leaf enzyme involves a sequential mechanism in which ATP and Pi react to give an enzymeP (E-P) intermediate (5). In the second partial reaction this intermediate apparently reacts with pyruvate to give PEP by a so-called “ping-pang” reaction, thus ATP+Pi+E 5 E-P + AMP + PPi
+ ATP + Pi =PEP
+ AMP + PPi
The enzyme was first demonstrated in the leaves of C4 plants (l), where it operates in mesophyll cell chloroplasts to catalyze the light-dependent conversion of pyruvate to PEP [see Ref. (2)]. It also occurs in various bacteria [see Ref. (3)] and has more recently been shown to occur in some other plant tissues [see Ref. (4)]. Various physical and kinetic properties of C4 leaf pyruvate,Pi clikinase have been examined using partially purified enzyme
E-P + pyruvate
= E + PEP.
Surprisingly, catalysis by bacterial pyruvate,Pi clikinase proceeds by a different mechanism designated as Tri Uni Uni Ping Pong (7). This sequence involves a reaction with ATP to give E-PP plus AMP followed by a reaction of E-PP with Pi to give E-P plus PPi, followed by a reaction of E-P with pyruvate as described above.
’ To whom correspondence should be addressed. ’ Abbreviations used: PEP, phosphoenolpyruvate; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. 53
0003-9861/85 $3.00 Copyright All rights
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
54
JENKINS
AND
We have recently described a complex mechanism for reversibly inactivating maize leaf pyruvate,Pi dikinase which accounts for the light-dark regulation of this enzyme in leaves (8, 9). This involves inactivation by ADP-dependent phosphorylation of an enzyme threonine residue and reactivation by phosphorolytic cleavage of this ester bond. These reactions depend on the existing phosphorylation status of pyruvate,Pi dikinase at its catalytic site and are controlled in various ways by substrates or products of this reaction. In view of these findings we sought more details about some of the properties of the maize leaf enzyme. We also used additional procedures to confirm that the maize pyruvate,Pi dikinase has a similar mechanism of catalysis to the one described earlier (see above) for the enzyme from sugarcane. MATERIALS
AND
METHODS
Plant tissue and materials.Maize plants (Zeo mays, variety Dekalb XL 81) were grown in soil in a greenhouse maintained between 20 and 30°C. Biochemicals and reagent enzymes were obtained from Boehringer-Mannheim, Australia or Sigma Chemical Company. PEP carboxylase used for the assay of pyruvate,Pi dikinase was prepared essentially as previously described (lo), except that the active fractions from DEAE-Sepharose-6B were concentrated and then chromatographed on a 180-ml column of Sephadex G-200 to obtain fractions free of pyrophosphatase. Pur$cntion of pyruvate,Pi dikinase. Pyruvate,Pi dikinase was extracted from maize leaves and then fractionated by precipitation with (NH&SO4 and Sephacryl S-300 column chromatography as previously described (ll), except that 5 mM dithiothreitol was used instead of 50 mM 2-mercaptoethanol and solutions were maintained anaerobic with nitrogen. The most active fractions from Sephacryl S-300 were pooled and then chromatographed on a DEAESepharose-6B column essentially as described by Burnell and Hatch (12). Peak fractions were at least 70% pure as judged by electrophoresis on dissociating acrylamide gels [see Ref. (8)]. Fractions free of pyrophosphatase were obtained by this procedure although the complete removal of this enzyme was not achieved in all preparations. Active fractions were stored at 2°C as a suspension in 65% saturated (NH,)zSOI solution. Alternatively, protein concentrated by precipitation with (NH&SO4 and then
HATCH
treated on a Sephadex G-25 column to remove (NH&SO, was stored at -20°C after adding an equal volume of glycerol. Assay of pllrmvate,Pi dikinase. All assays were conducted at 25°C. For the forward direction (pyruvate to PEP) the routine method was based on the spectrophotometric measurement of NADH oxidation associated with conversion of PEP to oxaloacetate (via PEP carboxylase) followed by reduction to malate via NAD-malate dehydrogenase (5). Reactions (1 ml) contained 25 mM Hepes-KOH, pH 8.0 (or pH 7.4 where specified), 8 mM MgSO,, 10 mM dithiothreitol, 10 mM NaHCOa, 1 mM glucose-6-P, 5 mM (NH1)zSOl, 2 mM pyruvate, 1 mM ATP, 2.5 mM potassium phosphate, 0.2 mM NADH, and about 0.25 unit of PEP carboxylase plus 1 unit of NAD-malate dehydrogenase. Generally, reactions were started by adding pyruvate,Pi dikinase. Where PEP inhibition of the forward reaction was examined activity was measured as AMP produced in reactions of a composition described above except that pyruvate was 0.5 mM, ATP was 0.4 mM, and NaHCOa, glucose-6-P, NADH, and the coupling enzymes were omitted. These reactions were incubated with varying concentrations of PEP and then stopped by adding HCl to give a final concentration of 0.025 M, and then stored at 0°C if they were to be immediately processed or frozen at -20°C. Samples were neutralized with KOH and then AMP was measured by following the total change of absorbance at 340 nm in reactions containing ATP, PEP, adenylate kinase, pyruvate kinase, NADH, and lactate dehydrogenase. These reaction mixtures for measuring AMP were essentially the same as described earlier for the assay of adenylate kinase (13). Pyruvate and any traces of ADP present in the samples were first consumed by mixing the sample with the reaction mixture from which adenylate kinase had been omitted. The absorbance change following the addition of adenylate kinase was then determined. For the reverse reaction (PEP to pyruvate) the assay was based on the previously described procedure (5) in which pyruvate production was measured spectrophotometrically by the NADH oxidized during conversion of pyruvate to lactate via lactate dehydrogenase. Reactions contained 25 mM Hepes-KOH, pH 7.4 (or 25 mM Tris-HCl, pH 8.0 were specified), 6 mM MgSO,, 10 mM dithiothreitol, 25 mM NH&l, 1 mM PEP, 0.5 mM AMP, 1 mM PPi, and 2 units of lactate dehydrogenase. Reactions were usually started by the addition of pyruvate,Pi dikinase. For ‘some experiments the rate of absorbance change at 340 nm observed with all reactants except AMP was subtracted from the rate observed with the complete reaction. Since pyruvate,Pi dikinase had a very high affinity for AMP the experiments conducted to determine
C4 LEAF
PYRUVATEJ’,
its K,, and others designed to determine the kinetics of interaction between AMP and PPi, employed an assay procedure which measured the conversion of [‘%]AMP to ATP. Reactions were as described above except that the PPi concentration was 0.5 mM and the [‘%]AMP concentration was varied. Reactions of 0.2 ml volume were started by adding 20 pl of pyruvate,Pi dikinase, previously diluted to be equivalent to an activity of about 0.0003 absorbance unit change min-’ for 20 pl of enzyme in a l-ml reaction measured by the spectrophotometric assay at 340 nm. Reactions were stopped at intervals by adding 20 pl of 2 M trifluoroacetic acid and stored on ice or at -80°C. Notably this acid is more volatile than water so that acidity does not increase when samples were subsequently spotted on thin-layer chromatogram plates. Samples of these reactions were spotted on polyethyleneimine-cellulose thin-layer plates, dried, and then developed in a solvent containing 1 M LiCl and 0.5 M acetic acid. Prior to spotting the samples on chromatograms the acidified reactions were supplemented with additional unlabeled AMP and ATP to facilitate subsequent detection of these compounds by viewing the developed chromatograms under ultraviolet light. The areas containing ATP and AMP were cut out and counted by the liquid scintillation procedure. Rates were calculated from the percentage conversion of [%]AMP to ATP. Reactions con[“CjAMP-ATP exchange react&n. tained 40 mM Hepes-KOH of varying pH, 7 mM dithiothreitol, 7 mM MgCl*, 5 mM NH&l, 0.66 mM ATP, and 0.16 mM [r4C]AMP, in a total volume of 0.3 ml. Orthophosphate, PPi, and PEP were added at the concentrations specified in Table III. Reactions were started by the addition of enzyme and then incubated at 25°C for intervals before stopping by the addition of 20 pl of 2 M trifluoroacetic acid and stored at -20°C. Rates of exchange were measured by determining the label appearing in ATP by chromatography as described above. Determining monovalent metal ion requirements. For studying the response to monovalent inorganic cations the following procedure was adopted to free reactions of cations present in reagents. Forward and reverse direction assay mixtures were made up except that enzymes, buffer, NADH, NaHC03, NH4Cl, MgS04, and, in the case of the reverse assay, AMP were omitted. The mixtures were passed through a 4-ml column of Dowex-50 (H+ form) to remove all cations and then 2 M Tris was added to adjust the mixtures to the desired pH (7.4 or 8.0). For the forward direction assay the following were then added to complete a reaction of 1 ml: 25 ~1 of 0.2 M MgS04, 20 al of 10 mM barium NADH, and 50 (~1of 0.1 M triethylamine bicarbonate, and then PEP carboxylase-malate dehydrogenase and also pyruvate,Pi dikinase previously treated on a Sephadex G-25
55
DIKINASE
column equilibrated with 10 mM Tris-HCl, pH 7.5, containing 5 mM MgSOl and 5 mM dithiothreitol. This treatment was designed to remove NH: and other monovalent metal ions from the enzyme preparations. A similar procedure was adopted for the reverse direction assay except that 25 ~1 of 0.2~ MgS04, 10 ~1 of lactate dehydrogenase (Sephadex G-25 treated), and 20 ~1 of 10 mM barium NADH were added before the pyruvate,Pi dikinase.
RESULTS
AND
DISCUSSION
Substrate Afinities and Product Inhibition Apparent Michaelis constants were determined for pyruvate, ATP, and Pi at pH 8.1 and 7.4, and for PEP, AMP, and PPi at 7.4 (Fig. 1 and Table I). The Lineweaver-Burk double-reciprocal plots shown in Fig. 1 for the forward direction substrates, pyruvate, ATP, and Pi, are for pH 8.1 only. All plots were linear. The values obtained for the forward reaction substrates differ substantially from some of those reported in earlier studies [90 PM for ATP at pH 8.3 (l), 250 PM for pyruvate, 15 PM for ATP, and 1.5 mM for Pi at pH 7.5 (S)]. Reverse reaction K, values for PEP and PPi were in close agreement with those reported earlier for the enzyme from sugarcane (15) and maize (6). The Km for AMP is very low and was not determined previously. We obtained values of 1.1 PM at pH 8.0 and 0.85 PM at pH 7.4. This may mean that the enzyme always exists as the AMP complex in vivo. Inhibition of the forward reaction by all three products, PEP, AMP, and PPi, briefly reported earlier (5), was examined in more detail. The data showing the interactions of PEP with pyruvate, of AMP with ATP, and of PPi with Pi, are shown in the form of Linewaver-Burk reciprocal plots in a later section of this paper (see Fig. 4). The results for Ki values and types of inhibition are summarized in Table I. For PEP the Ki was 150 PM and inhibition was linearly competitive with respect to pyruvate. The Ki was 130 PM for AMP and 320 PM for PPip and inhibition was noncompetitive with respect to ATP and Pi, respectively.
56
JENKINS
h:
6
,z E c 0 ”
4
5
2
0
0
6
12
l/Pyruvate
l/PEP
18
0
(mM”)
(mM-‘)
AND
8
HATCH
16 1 /ATP
(mM-‘)
l/AMP
(PM-‘)
24
I
I
I
1
I
1
0
1
2
3
4
5
~/PI (mM-‘J
FIG. 1. Lineweaver-Burk plots for the substrates of pyruvate& dikinase in the forward and reverse directions. For the forward direction (substrates: pyruvate, ATP, and Pi) the pH was 8.0 and for the reverse direction the pH was 7.4. Other details of assay procedures and reaction mixture composition are provided under Materials and Methods.
TABLE
Requirement for Monovalent Cations
I
Km VALUESFOR SUBSTRATESOF PYRUVATE, Pi DIKINASEANDTHE Ki VALUESFOR PRODUCT INHIBITORS Km (~4 Substrate
pH 8.1
Forward reaction Pyruvate ATP pi
82 32 380
Reverse reaction PEP AMP PPi
-
1.1
Inhibitor
K (/.M
Forward reaction PEPa AMP PPi
150 130 320
pH 7.4
92 48 560 160 0.85 46
(C versus pyruvate)* (NC versus ATP) (NC versus Pi)
EPEP inhibition was mixed (noncompetitive) with respect to both Pi and ATP. “C and NC refer to competitive and noncompetitive inhibition, respectively.
Maize leaf pyrllVRk?~i dikinase requires the divalent metal ion Mg2+ for activity (1); at least for reverse direction activity a monovalent cation is also essential although only marginal stimulation of the forward reaction was reported (6). We further investigated these requirements. Using assay mixtures treated to remove metal ions associated with reagents we showed that both the forward and reverse directions of the pyruvate,Pi dikinase reaction are almost totally dependent on added monovalent cations. For the forward direction reaction assayed at pH 8.1 this requirement was satisfied by relatively low concentrations of NH:, or much higher K+; Na+ was much less effective (Fig. 2). For the reverse direction much higher NH: was required for maximum activity; there was a partial response to K+ but no effect of Na+. Table II summarizes the responses to NH:, K+ and Na+ seen in Fig. 2 and also includes data for the forward reaction at pH 7.4; also recorded are K, values where responses
Cd LEAF
PYRUVATE,Pi
Catlo”,rnM,
cmon,rnM,
FIG. 2. Monovalent cation requirements for pyruvate,Pi dikinase activity in the forward (pyruvate to PEP) and reverse directions. Reactions were treated to remove monovalent metal ions as described under Materials and Methods. All cations were provided as their chloride salts.
were significant. The K, values for K+ in the forward direction (determined from double-reciprocal plots) were about 30 times those for NH: (Table II). Notably, the K, value for NH: for the reverse reaction was about 10 times that for the forward reaction. In other experiments we confirmed an earlier observation (1) that the divalent metal ion requirement for the forward reaction was satisfied by a Mg2+ concentration of about 3 mM in excess of the TABLE
DIKINASE
57
ATP concentration. Additional Mg2+ up to 50 mM had no effect in reducing the requirement for NH: or other monovalent metal ions. These data on monovalent metal ion requirements are similar to those obtained for bacterial pyruvate,Pi dikinase (14). However, our results differ quantitatively from those reported by Sugiyama (6) for metal ion responses of the reverse reaction catalyzed by maize leaf enzyme. He recorded a K, of about 5 mM for NH: at pH 7.4 and obtained much higher degrees of activation with K+ and also Na+. Although NH: is the most effective activator of pyruvate,Pi dikinase in vitro it may not contribute to the activation of this enzyme in vivo. We found that the K, for NH: was about 0.3 mM for the forward reaction and much higher for the reverse reaction. However, estimates of metabolic compartment NH: concentrations in vivo, based on NH3 exchange with leaf tissue, would indicate concentrations of only about 30 j&M NH: at pH 7.0 (15). At diffusion equilibrium this concentration would be even less at pH 8, about the presumed pH of the chloroplast stroma in the light (16). However, at least the forward reaction for pyruvate,Pi dikinase is equally effectively activated by II
MONOVALENT METAL ION REQUIREMENT FOR PYRUVATE, Pi DIKINASE Activation
Cation and pH Forward reaction NH:, pH 8.1 KC, pH 8.1 Na+, pH 8.1 NH:, pH 7.4 K+, pH 7.4 Na+, pH 7.4 Reverse reactionb NH;, pH 7.4 K+, pH 7.4 Na+, pH 7.4
(Pyruvate
response
Ka Cm@
to PEP) >20-fold >20-fold ~25% activated by 40 InM” >20-fold >20-fold About 15% activated by 40 >20-fold About 40% activated No effect
DPercentage activation refers to the activity relative to the maximum Concentrations above about 80 mM K+ or Na+ inhibit activity. bAt pH 8.1 the reverse reaction is activated only about seven-fold essentially no effect.
by 40
0.38 10 0.25 8 -
mM”
2.0 10 -
mMa
observed with optimal by NH:
while
NH;
or KC.
K+ and Na+ have
58
JENKINS
K+ as by NH: although concentrations of up to 80 mM K+ are required for full activity. This presumably presents no problem in viva since the natural K+ concentration in chloroplasts is estimated to be about 100 InM (17).
Efect of Varying pH Although leaf pyruvate,Pi dikinase has been studied in some detail (1, 5, 6) little information has been reported about the effect of varying pH on the forward direction and none is available for the reverse direction. As previously reported (1) the optimum for the forward reaction was about 8.2; there was very little activity below pH 7 (Fig. 3). Reverse direction activity was optimal at about pH 6.8 and activity declined on the alkaline side of this pH so that there was only about 10% of maximum activity at pH 8.4. Activities for the forward and reverse reactions were about equal at pH 7.6. The marked opposite effects on the forward and reverse reactions of increasing pH in the range from about 6.8 to 8.3 are probably related to the fact that the forward reaction results in a stoichiometric production of 2H+ for each pyruvate converted to PEP (18). In C4 leaves pyru-
AND
HATCH
vate,Pi dikinase operates in mesophyll chloroplasts in the direction of PEP synthesis (2). Notably, activity will be optimal for this direction at the predicted pH of about 8.2 for the stroma in the light (see above), while the reverse reaction activity will be relatively low. It may also be significant that the likely NH: levels in chloroplasts will be far too low to activate pyruvate,Pi dikinase in this direction (see above discussion). Since K+ only partially activates the reverse reaction and Na+ not all, the combination of this limited activation and the high stromal pH may render activity in the direction of pyruvate synthesis insignificant in vivo.
Reaction Mechanism The following experiments were conducted to test the validity of an earlier conclusion (5) that the leaf pyruvate,Pi dikinase proceeds by a two-step mechanism (see Introduction) rather than the three-step mechanism reported for the bacterial enzyme (7). For the leaf enzyme the mechanism proposed [Bi Bi Uni Uni according to the nomenclature of Cleland, Ref. (19)] is E + ATP + Pi * E-P + AMP + PPi E-P + pyruvate
+ E + PEP.
[Al
The mechanism proposed for the bacterial enzyme (Tri Uni Uni) is E + ATP = E-PP + AMP E-PP + Pi C= E-P + PPi E-P + pyruvate
7.0
7.5
80
85
PH
FIG. 3. Effect of varying pH on the activity of pyruvate,Pi dikinase in the forward (pyruvate to PEP) and reverse directions. All reactions were buffered with a 20 mM Hepes-20 mhl Tricine mixture adjusted with KOH to the desired pH. The actual pH of reactions were measured after assays were completed.
[Bl
c E + PEP.
A critical reaction for distinguishing between these mechanisms is the partial reaction leading to [14C]AMP-ATP exchange. Predictions are that Pi and PPi will be required for the AMP-ATP exchange if mechanism A is operative [see the above scheme and Refs. (5, 7)]. With mechanism B there will be no other requirements but Pi or PEP will inhibit due to conversion of the enzyme to the E-P form, which does not participate in this particular exchange reaction. We con-
Cd LEAF
PYRUVATE,P,
firmed our earlier observation (5) that with the leaf pyruvate,Pi dikinase this [14C]AMP-ATP exchange is stimulated several fold by the combined addition of Pi and PPi (Table III). Increases of UP to sixfold were observed although the response varied between experiments and tended to be less at lower pH. Although reagents were treated to remove contaminating Pi and PPi (see footnote, Table III), traces of these compounds may have contributed to some of the activity seen in the control reactions. However, the fact that there was usually little response to adding either Pi or PPi separately supported the view that there was little contamination by Pi and PPi in the control reactions (Table III). In this respect the exception was the substantial increase seen by adding Pi alone at pH 7.0. A similar response to Pi was seen in a separate experiment not quoted here, but we have no simple explanation for this TABLE
III
REQUIREMENT FOR CATALYSIS EXCHANGE
REACTION
OF [%]AMP-ATP BY PYRUVATE, Pi DIKINASE
Exchange activity (rmol ml-’ 4 min-‘) Expt. 1
Expt. 2
Additions to basic reaction”
PD 8.0
PD 7.5
PH 8.2
PD 7.0
None pi PPi Pi + PPi Pi + PPi + PEPb PEP
0.77 0.91 0.77 2.9 3.5 0.77
0.70 0.91 0.73 2.0 5.2 0.81
0.38 0.38 0.35 2.4 3.2 0.38
0.38 1.2 0.33 2.3 9.1 0.42
“Pi concentration was 1 mM for Expt. 1 and 2.5 mM for Expt. 2. Concentrations of PPi and PEP were 0.08 and 1 mM, respectively. For these experiments the ATP used was treated on a Sephadex G10 column to remove Pi and PPi and the Pi stock solution was chromatographed on DEAE-Sephadex (8) to remove PPi. b This reaction contains all reverse direction substrates (PEP, AMP, and PPJ and hence measures reverse reaction activity.
DIKINASE
59
result. Notably, the rate of the [14C]AMPATP exchange was close to the rate of the reverse reaction activity (given by reactions supplemented with PPi plus PEP) at about pH 8.0 but was much less than the reverse reaction rate at lower pH. Presumably this is due to increasing reverse reaction activity as the pH is reduced (see above and Fig. 3). This increase may not be accompanied by increased exchange reaction activity due to the constraint imposed by the forward reaction. The origin of the AMP-ATP exchange activity seen in the control reactions containing no added Pi or PPi (see Table III) is uncertain. As discussed above, if this was due solely to contaminating Pi and PPi then larger responses to adding Pi or PPi alone might have been expected. On the other hand, if this activity was due to the first partial reaction of mechanism B then Pi and PEP would be expected to inhibit (see above) and no such inhibition was observed (Table III). As discussed in detail by Milner and Wood (7) the alternative reaction mechanism for pyruvate,Pi dikinase may also be distinguished by the kinetics of inhibition of the forward reaction by the products, PEP, AMP, and PPi, and by the kinetic patterns resulting from varying certain substrate pairs. We found that with the leaf pyruvate,Pi dikinase PEP inhibited competitively with respect to pyruvate and that AMP and PPi inhibited noncompetitively with respect to ATP and Pi, respectively (Fig. 4). The results for AMP and PPi inhibition are in accordance with the predictions outlined by Milner and Wood (7) for mechanism A. They are not consistent with the operation of mechanism B, where competitive inhibition is predicted. Different patterns of inhibition of the forward reaction by PEP have been predicted depending on whether the partial reactions of mechanism A, and also of mechanism B, occur at a common overlapping site or at functionally independent sites [see Ref. (7)]. Our observation that PEP inhibits competitively with respect
60
JENKINS
OmM
o0
AND
HATCH
PEP
-1
2 1 /Pyruvate
3
4 (mM-‘)
5
0
4
8 1 /ATP
12
160
1
2
3
l/Pi
(mM”)
4
5
(mM”)
FIG. 4. Kinetics of the inhibition of the forward reaction of pyruvate,Pi dikinase by the products, PEP, AMP, and PPi. The pH was 8.0 and other details of assay procedures are described under Materials and Methods.
to pyruvate (Fig. 4) indicates that the second partial reaction of mechanism A (pyruvate-PEP exchange) takes place at a site which is functionally independent of the one catalyzing the first partial reaction. Further support for this alternative was provided by the observation that PEP inhibited noncompetitively with respect to ATP and Pi (results not shown). As discussed by Milner and Wood (7), there are only two substrate pairs which interact to give kinetics that distinguish mechanism A from mechanism B. These are AMP-PPi and ATP-Pi. For the former pair the Lineweaver-Burk reciprocal plot we obtained when both AMP and PPi were varied gave intersecting lines (Fig. 5). This agrees with the operation of mechanism A; mechanism B should give parallel lines. The prediction for the plots obtained when ATP and Pi are simultaneously varied are for intersecting lines with mechanism A and parallel lines with mechanism B. The data we obtained in several experiments consistently gave plots showing essentially parallel lines (Fig. 5), a result apparently not consistent with the operation of mechanism A. This pattern indicates that ATP and Pi interact to affect the binding of each other to the enzyme, with affinity increasing as the concentration of the other substrate decreases (see Fig. 5). This may be explained if mechanism A operates but the binding of ATP and Pi to free enzyme is higher
than the binding to the Es Pi and E * ATP forms, respectively. CONCLUDING
COMMENTS
Maize leaf pyruvate,Pi dikinase operates in the light in chloroplasts to convert pyruvate to PEP. For spinach chloroplasts at least, the pH in the stroma varies between about pH 7.2 in the dark and about pH 8.2 in the light (16). Clearly, the pH response for the forward reaction of pyruvate,Pi dikinase (see Fig. 3) ideally suits this enzyme for this particular role. As noted above, the decreasing reverse reaction activity as pH increases, and the likelihood of only partial activation of the
“0
10
20
IlnTPlmM’)
30
0
1
2 ,/AMP
3
4
#o.v’I
FIG. 5. Kinetic responses to simultaneously varying the substrate pairs ATP and Pi for the forward reaction and AMP and PPi for the reverse reaction assessed by Lineweaver-Burk reciprocal plots. The pH was 8.0 and other details are provided under Materials and Methods.
Cd LEAF PYRUVATEQi
reverse reaction by monovalent metal ions in viva (see above) would combine to make activity in this direction negligible in the light. Other evidence would suggest that these particular pH-activity responses were probably not evolved especially to optimize the operation of this particular enzyme in this particular situation. For instance, pyruvate,Pi dikinase from bacteria, including the enzyme from Bacteroides symbioses which apparently operates to convert PEP to pyruvate in viva, have similar pH response characteristics. Like the maize leaf pyruvate,Pi dikinase these bacterial enzymes are also more active in the reverse direction than the forward direction when each activity is determined under optimal conditions. As already discussed, it appears most likely that the pH responses are largely a consequence of the involvement of H+ in the reaction. There appears to be no obvious significance in the differences in K, for the substrates of pyruvate,Pi dikinase from different sources. There were substantial differences in some of the K, values for pyruvate, ATP, and Pi with enzyme from such diverse sources as maize leaf, wheat seed (2), and various bacteria [see Ref. (3)], all of which operate in viva for PEP synthesis. Notably, the K, values for substrates in the B. symbiosis enzyme [believed to operate in the reverse direction in viva; see Ref. (21)] fell within the range of values recorded for the other enzymes. The K, for AMP for all these enzymes is very low (range, 1-15 PM). The only explanation we can offer for this is that strong binding of AMP may increase catalytic efficiency through the effect described by Albery and Knowles (22) of reducing the free energy difference between kinetically critical intermediate forms of the overall reaction. Earlier studies (5) of exchange reactions catalyzed by the sugar-cane leaf pyruvate,Pi dikinase indicated a Bi Bi Uni Uni mechanism (referred to above as mechanism A). Later evidence [see Ref. (7)] indicated that bacterial pyruvate,Pi dikinase operated by a Tri Uni Uni mechanism
61
DIKINASE
(mechanism B described above). Our present studies confirmed by various additional procedures the operation of the A-type mechanism for the maize leaf enzyme. The significance of this difference remains to be resolved. One possibility is that the A-type mechanism may be important for the processes leading to the light-dark regulation of pyruvate,Pi dikinase. For this complex process only pyruvategidikinase phosphorylated at the catalytic-site histidine is inactivated by ADP-mediated phosphorylation at a separate site on the enzyme; by contrast, Pimediated activation by phosphorolytic dephosphorylation is most rapid with the noncatalytically phosphorylated form of the enzyme (8, 9, 23). Notably, inactive pyruvate,Pi dikinase does not catalyze the pyruvate-PEP exchange reaction and hence cannot be dephosphorylated at its catalytic site by pyruvate (23); however, this inactive enzyme does catalyze the first exchange reaction between ATP + Pi and AMP + PPi (see mechanism A above). Consequently, AMP + PPi can react with the inactive enzyme to remove the catalytic-site phosphate group and hence convert it to a form that is more rapidly activated by a reaction with Pi. ACKNOWLEDGMENT We acknowledge the expert technical assistance of Tony Agostino. REFERENCES 1. HATCH, M. D., AND SLACK, C. R. (1968) &o&em J. 106, 141-146. 2. HATCH, M. D., AND OSMOND,C. B. (1976) in Encyclopedia of Plant Physiology (New Series) (Stocking, C. R., and Heber, U., eds.), Vol. 3, pp. 144-184, Springer-Verlag, Heidelberg. 3. WOOD,W. A. (editor) (1975) in Methods in Enzymology (Wood, W. A., ed.), Vol. 42, pp. 18’7212, Academic Press, New York. 4. EDWARDS,G. E., NAKAMOTO,H., BURNELL,J. N., AND HATCH, M. D. (1985) Annu. Rev. Plant Physiol, in press. 5. ANDREWS, T. J., AND HATCH, M. D. (1969) Biochem. J. 114, 117-125. 6. SUGIYAMA,T. (1973) Biochedst?y 12,2862-2868. 7. MILNER, Y., AND WOOD, H. G. (1976) J. Bid Chem 251, 7920-7928.
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8. ASHTON, A. R., BURNELL, J. N., AND HATCH, M. D. (1984) Arch. Biochem. Biophys. 230,492503. 9. BURNELL, J. N., AND HATCH, M. D. (1984) Arch Biochem. Biophys. 231,1’75-182. 10. HATCH, M. D., AND HELDT, H. W. (1985) AnaL Biockem., in press. 11. HATCH, M. D. (1979) Aust. J. Plant Physid 6, 60’7-619. 12. BURNELL, J. N., AND HATCH, M. D. (1985) Arch Biochem. Biqphys., in press. 13. HATCH, M. D. (1982) Aust. .I Plant Physid 9, 287-296. 14. MICHAELS, G., MILNER, Y., MOSKOVITZ, B. R., AND WOOD, H. G. (1978) J. BioL Chem. 253, 76567661. 15. FARQUHAR, G. D., FIRTH, P. M., WETSELAAR, R., AND WIER, B. (1980) Plant PhysioL 66, 710714.
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16. HELLIT, H. W., WERDEN, K., MILOVANCEV, M., AND GELLER, G. (1973) Biochem Biophys. Ada 314,224-241. 17. DEMMIG, B., AND GIMMLER, H. (1983) Plant Physiol. 73,169-174. 18. REEVES, R. (1968) J. Bid
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19. CLELAND, W. W. (1970) in The Enzymes (Boyer, P. D., ed.), Vol. 2, pp. l-65, Academic Press, New York. 20. MEYER, A. O., KELLY, G. J., AND LATZKO, E. (1982) Plant PhysioL 69, 7-10. 21. WOOD, H. G., O’BRIEN, W. E., AND MICHAELS, G. (1977) A&J. EnzymoL 45, 85-155. 22. ALBERY, W. J., AND KNOWLES, J. R. (1976) Biochemistry 15,5631-5640. 23. BURNELL, J. N. (1984) B&hem. Commun 120. 559-565.
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