ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 191, No. 2, December, pp. 657-665, 1978
Hysteretic
Kinetic Behavior of Beef Liver Pyruvate Carboxylase’ BERWIN
P. YIP2 AND FREDERICK
B. RUDOLPH
Department of Biochemistry, Rice University, Houston, Texas 77001 Received February
22, 1978; revised July 27, 1978
Beef liver pyruvate carboxylase can exhibit transients of enzyme activity depending on prior incubation conditions. At pH 7.0, when the reaction is initiated with MgATP (after incubation with the other reaction components) an initial burst of activity occurs which decays to a slower rate. If the reaction is initiated by addition of enzyme, acetyl-CoA, or pyruvate, a lag occurs followed by an increase in activity to a steady state level after about one to two minutes. The lag transient has been analyzed by a pseudo fast order rate equation derived by Hatfield et al. (J. Biol Chem. 245, 1748-1754, 1970) assuming a slow transition from one enzyme conformation to another. The rate constant for the transition was independent of enzyme concentration and MgATP and bicarbonate concentrations in the range tested, but was markedly dependent on pyruvate concentration. From the temperature dependence of the rate of transition, a value of 1.7 kcal was calculated for the conformational change. At pH 8 with additional salt (0.1 M KCl), the transient effect was diminished but can still be observed in some cases. It is suggested that biphasic double reciprocal plots observed with pyruvate may be due to a pyruvate-induced conformational change to a more active form at high pyruvate levels.
Pyruvate carboxylase (pyruvate:COz ligase (ADP forming), EC 6.4.1.1) catalyzes the reaction:
two classes of vertebrate pyruvate carboxylase. The enzymes isolated from chicken liver, turkey liver (l), and sheep kidney (2) are similar as are the ones from rat (3) and calf liver (1). During studies on the beef liver pyruvate carboxylase, a lag in the assay was repeatedly observed when the reaction was initiated by the addition of the enzyme. A few previous investigations with pyruvate carboxylase from various sources have mentioned such phenomena (4), but no studies were carried out to characterize it. This report describes the results of a detailed investigation into this effect. The possible metabolic significance of this “hysteretic” (5) behavior is discussed.
Pyruvate
+ MgATP2acetyl-CoA + HCOS- \ ’ oxalacetate Mg2+, K+ + MgADP- + Pi.
This reaction serves a gluconeogenic role in liver, kidney, and some microorganisms and an anaplerotic role in other tissues and species. It has been suggested that many factors are involved in its regulation (1). The enzyme has been studied from various sources and is a tetrameric protein containing bound metals and needing several activators (1). By the criteria of sensitivity to cold inactivation, immunochemical studies, and relationship between the quaternary structure and activity, there appear to be
MATERIALS
AND
METHODS
Beef liver pyruvate carboxylase was purified using a modification of the procedure developed for the rat liver enzyme (3). The lyophilized mitochondrial powder was extracted with 0.05 M KPi buffer, pH 7.0, containing 5 mM ATP, 5 mM MgS04 and 0.5 mu EDTA, followed by a 28-35% saturation of ammonium sulfate precipitation. The heat step described in (3) was omitted. The precipitate was dissolved in 0.1 M Tris-Cl buffer at pH 7.2 containing 0.1 mM EDTA, 1
’ This research was supported in part by Grant C582 from the Robert A. Welch Foundation and Grant CA 14030 from the National Cancer Institute, DHEW. ’ Recipient of a Robert A. Welch Foundation Predoctoral Fellowship. Present address: Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin. 657
0003-9861/78/1912-0657$02.00/O Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.
658
YIP AND
mu dithiothreitol and 12.6 g/100 ml (NH&S04. After centrifugation to separate the precipitate, solid ammonium sulfate was added to the supernatant to a concentration of 50 g/100 ml and the precipitate obtained was extracted successively as described in (3). The 14.4 and 12.6 g of ammonium sulfate/100 ml fractions were pooled and the protein was precipitated by adding solid ammonium sulfate to the pooled fractions to a final concentration of 50 g/100 ml. The precipitate was dissolved in a minimal volume of 0.1 M Tris-Cl, 1 mu EDTA, 1 mM dithiothreitol, pH 7.2 and applied to a Sepharose 6B column (2.4 x 25 cm) previously equilibrated with the same buffer. The fractions containing the enzyme activity were pooled and concentrated by ammonium sulfate precipitation. The specific activity at pH 8.0 was 6-10 units/mg protein. Attempts were also made to purify the enzyme further by DEAE column chromatography as described in (6) and on sucrose gradients (lo-258 linear gradient). The specific activity increased somewhat but the protein obtained was not homogeneous based on polyacrylamide gel electrophoresis. The enzyme obtained from the Sepharose column did not show any contaminating enzyme activity which would interfere in the enzyme assays, and was routinely used in most experiments. In addition, enzyme processed through additional DEAE chromatography and sucrose gradient purification steps exhibited the same transients as the enzyme from the Sepharose column. The concentrated enzyme, which was stored in sucrose buffer (3), was dialyzed overnight against 0.1 M Tris-Cl, 0.1 mru EDTA and 1 mM dithiothreitol, pH 7.2, before use. The materials were obtained from the following indicated sources: acetyl-CoA, dithiothreitol, NADH, Sigma; ATP, Calbiochem; pyruvic acid, J. T. Baker; Na[‘%]HC03, Amersham/Searle; [Y-~‘P]ATP, tetra(triethylammonium) salt, New England Nuclear; Sepharose 6B, Pharmacia. Assays. The enzyme was routinely assayed by the malate dehydrogenase coupling enzyme assay as described previously (3). pH was maintained at pH 7.0 in 0.1 M Hepes3 buffer. The reactions were monitored in a thermostated Cary 118 spectrophotometer. The concentrations (mM) of the assay mixture, in a total volume of 1 ml, were: pyruvate, 5; KHCOJ, 20; MgATP, 0.8; acetyl-CoA, 0.16; NADH, 0.16. The components were incubated at 30°C prior to initiation of the reaction. The incorporation of radioactive carbon dioxide into oxalacetate was assayed as described in (7) with the following modifications. The reaction mixture contained, in a total volume of 0.25 ml, the following mu concentrations of substrates: Hepes, 100, pH 7.0; pyruvate, 5; KHCO,; 20, MgATP, 2.4; acetyl-CoA, 0.16; ’ Abbreviations used: Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; TCA, trichloroacetic acid.
RUDOLPH [“C]HC03-, 3.3 (sp. act. 60.2 mCi/mmol). A lo-p1 sample of the reaction was transferred at timed intervals (usually 10 s for fit 10 samples and 20 s for next 10 samples) into 50 )d of 3.6% cold TCA in a small disposable centrifuge tube, followed by thorough mixing. The samples were neutralized with KOH. Malate dehydrogenase and NADH (final concentration, 0.3 mM) were then added to the mixture to convert the oxalacetate to malate. After 10 min at room temperature, 50 al of 10% cold TCA was added, and the mixture was centrifuged (1006 rpm). Residual radioactive carbon dioxide was driven off by bubbling with carbon dioxide for 10 min. Aliquots (100 ~1) were removed for determination of [‘%]malate formed. They were counted in a liquid scintillation spectrophotometer using a scintillation fluid with the following composition (toluene, 2100 ml; ethanol, 1230 ml, 2,5-diphenyloxazole, 12 g; 1,4-bis[2-(5phenyloxazolyl)]benzene, 0.3 g). The reaction was also followed by determination of the “Pi released from [y-32P]ATP. The assay mixture contained in a volume of 0.25 ml, the following mM concentrations: pyruvate, 5; acetyl-CoA, 0.16; KHCO$, 20; MgATP, 0.16; Hepes, 100, pH 7.0 and [y-3ZP]ATP, 8.9 X lOma (sp. act. 28.2 Ci/mmol). A lo-$ aliquot of the reaction mix was transferred at timed intervals into 50 al of 6% cold TCA in a small centrifuge tube. One hundred microliters of isobutanol; benzene (Hz0 saturated) (1:l) was added and the solution vortexed for 15 s. Ten microliters of a 40 mM molybdate solution (in 4 N H&04) was then added to each of the centrifuge tubes and the mixture was vortexed vigorously for 30 s. The samples were then centrifuged for 2 mm (1006 rpm). Aliquots of the organic layer (50 )rl) were removed and counted in the medium described above. RESULTS
During routine assays in studies with beef liver pyruvate carboxylase, it was observed that, under some conditions, the reaction velocity changed with time. When the reaction was initiated with the enzyme or pyruvate, the reaction progress curve exhibited a lag, i.e., the reaction rate was slow at the beginning and increased over a period of a minute to a steady state rate. Bicarbonate initiated reactions also exhibited a lag period, but with a shorter time period prior to steady state attainment. A different effect was observed when the reaction was initiated with MgATP. The initial rate of the reaction exhibited a burst transient with the reaction starting at a fast rate and decaying slowly. In general, a steady state reaction was not observed with the MgATP initiated reaction. The coupled enzyme assay was used in
BOVINE
LIVER
PYRUVATE
‘i
FIG. 1. Radioactive assays of pyruvate carboxylase. Details are described in Materials and Methods. (0) [‘%]HC03incorporation; ATP initiated reaction. (0) [‘%]HC03incorporation; enzyme initiated reaction. (x) [32P]phosphate release; enzyme initiated reaction (a different amount of activity was used for this assay than for the other two).
initial studies and the lag effect could be attributed to the inherent lag in the coupling enzyme system. However, malate dehydrogenase was always present in excess, and additions of more coupling enzyme did not affect the observed lag. Independent methods of assay were used to confirm the validity of the assay, where the enzyme reaction was followed by measuring the amount of [‘4C]bicarbonate incorporated into oxalacetate and by the release of radioactive inorganic phosphate from [T-~‘P] ATP as shown in Fig. 1. The lag was also present when the reaction was initiated by addition of the enzyme and distinctly different from the transient observed when the reaction was initiated with MgATP. To quantitate the extent of the lag phase, the ratio (R,,f) of the initial reaction velocity (initial slow phase) to the steady state velocity (final fast velocity) was calculated (8). Initial velocity measurements were made about 18 sec. after the addition of the component to initiate the reaction, which was the time required for mixing and instrument set-up and thus do not measure the actual initial velocity. However, the R,,f values still provide a good estimate of the extent of the lag phase as measurements were made in a consistent manner. Under
CARBOXYLASE
659
TRANSIENTS
the standard usual conditions, when the reaction was initiated with the enzyme, the Rslfvaried within the range of 0.5 to 0.8 for different preparations of the enzyme. To evaluate the lag effect, a series of incubations with different combinations of substrates was done (Table I). The first three conditions are as described above. A MgATP initiated reaction is not listed because a steady state rate was not usually observed. However, by using the steady state rate obtained from the enzyme initiated reaction as the steady state rate for the MgATP initiated reaction, the R,,f is 1.5. Incubations 2, 3, and 5 illustrate that a lag is observed only when the enzyme is preincubated with MgATP. Enzyme preinTABLE
I
EFFECT OF INCUBATION WITH SUBSTRATES ON LAG PHASE Reaction 1. 2. 3. 4. 5. 6. 7. 8.
9. 10.d 11.’
initiated
with”
Enzyme Pyruvate HCOaATP + pyruvate HC03- + pyruvate ATP + HCOZEnzyme preincubated with dithiothreitol Enzyme preincubated dithiothreitol with and bovine serum albumin ATP + pyruvate + HCO,ATP + pyruvate + HCOaATP + pyruvate + HCOa-
K
V
0.036 0.050 0.051 0.104 0.047 0.080 0.038
0.066 0.067 0.065 0.104 0.072 0.080 0.070
0.54 0.75 0.78 1.00 0.66 1.00 0.54
0.048
0.086
0.56
0.062
0.062
1.90
0.081
0.081
1.00
0.092
0.092
1.06
k/f
a Assay was as described under Materials and Methods. Final enzyme concentration was the same for ah cases. The enzyme was incubated with ah the components except those to be added as indicated for 2 min at 30°C and the reaction was initiated with the component indicated. The numbers represent the average of at least four determinations. * V,, initial velocity in A OD340.,/40 s, was taken at the earliest time possible (usually after about 20 s of addition of enzyme or substrate). ’ V,, final steady state velocity in A ODsO .,/40 s, was measured 2 min after the start of the reaction, and was linear for at least 1 min. d Dithiothreitol was also present in the cuvette. ‘Dithiothreitol and bovine serum albumin were also present in the cuvette.
660
YIP AND
cubated with dithiothreitol(O.2 mu) or bovine serum albumin (0.5 mg/ml) also exhibited the lag transient. When the enzyme was added into the cuvette first (with buffer and acetyl-CoA) and the reaction initiated with all the substrates (70-g addition), no transient was observed and the reaction progress was linear with time for more than 2 min. Inclusion of dithiothreitol and bovine serum albumin did not have any effect on the linearity of the reaction progress, although inclusion. of these two reagents activated the enzyme slightly. Beef liver pyruvate carboxylase is functionally dependent on acetyl-CoA for activity. When acetyl-CoA is omitted from the assay mixture, and the enzyme is incubated with all the substrates and the coupling enzyme system, no reaction was detected. Addition of acetyl-CoA restores the activity and the reaction exhibited a lag phase (Fig.
RUDOLPH
between two enzyme species of different intrinsic activity. The transition can be analyzed according to the equation outlined by Hatfield et al. (9). A similar equation has also been described by Frieden (5). As illustrated in the inset of Fig. 2, graphical analysis of the data by this method yielded apparently linear first order log plots, suggesting that the analysis is descriptive of the phenomena observed. The pseudo-first order constant for the conversion of the less active enzyme species to the more active form, k’, can be obtained from the slope of the log plot (9). In this analysis, a knowledge of the true initial velocity was not
0.'
2).
The above studies were carried out at saturating levels of substrates. When the experiments were done with Km levels of substrates (i.e., 0.08 mu MgATP, 2 mu bicarbonate, and 0.5 mu pyruvate), a somewhat different behavior of the enzyme was observed. No lag was observed with pyruvate added last to initiate the reaction but a lag was still observed when the enzyme was used to initiate the reaction. When the reaction was studied by initiating with all three substrates, the reaction trace showed a burst transient, gradually slowing down. The burst was also observed when MgATP was used to initiate the reaction. This phenomenon was apparently not caused by substrate depletion at the lower substrate concentrations used, because the reaction progress initiated by pyruvate or enzyme still showed a linear rate when the same amount of reaction had taken place. The transient was dependent on substrate concentrations. Thus, in Fig. 3, when the MgATP concentration is markedly increased (arrow), during the steady state of a reaction in the presence of non-saturating MgATP, the reaction progress goes through another transient. Similar results were obtained with pyruvate as the varying substrate (data not shown). The transient indicates a slow transition
0.3
‘+%a
0.2
0.1
I
0
20
40 TIME
60
SO
lrccl
PIG. 2. Reaction progress curve generated by adding acetyl-CoA to initiate the reaction. The enzyme was incubated with all the reaction components minus acetyl-CoA. No reaction was detected. Acetyl-CoA was then added (10 $ addition; final concentration 0.13 mg/ml) to activate the enzyme. Inset: Analysis of lag phase involving a first order process as described by Hatfield et al. (9). A A, represents the maximum difference in absorbance change between an imaginary linear progress curve with the reaction velocity the sane as the steady state velocity of the transient and the progress curve exhibiting the lag. A At represents the difference between the two progress curves at time t of the lag phase. See Ref. (9) for details of the rate equation and a graphical illustration of the analysis. The pseudo-Srst order rate constant for the line shown
is 0.05 s-1.
BOVINE LIVER PYRUVATB 0.3
0.2 AA 3.0
0.1
0
50
100 TIME
150 (set
200
)
FIG. 3. Reaction progress curve with steady state addition of supplemental substrate. Curve A is obtained with 0.04 mre MgATP, the other substrate concentrations as described in Materials and Methods. At the srrow, MgATP was added to yield a final concentration of 0.34 mru (curve B). Curve C is that obtained with 034 mru MgATP, with MgATP added last. Curve D is that obtained with 0.34 mru MgATP with enzyme added last.
required. Subsequent calculations of the rate of transition were determined by linear regression using a digital computer. Since it was not possible to obtain steady state rates for MgATP initiated reaction, further studies were carried out only with enzyme or pyruvate initiated reactions. Saturating substrate concentrations were employed because one of the conditions for the equation derived by Hatfield et al. (9) was that the substrate concentration remained constant throughout the time course of the analysis. Effect of enzyme concentration. Progress curves generated by adding different amounts of the same enzyme preparation to initiate the reaction were analyzed and the pseudo-first order rate constants obtained were plotted versus the amount of the enzyme used (Fig. 4). The pseudo-first order rate constants stayed fairly constant over the enzyme concentrations used. The R,,f values obtained were also constant (0.65 f 0.05). Also, as shown in Fig. 4, the velocity obtained by taking the slope of the steady state portion of the progress curve was proportional to the concentration of
CARBOXYLASE
661
TRANSIENTS
the enzyme added. When the reaction was initiated with all three substrates added together after preincubation with acetylCoA, the reaction progress curves were linear, but the plot of the velocity versus enzyme concentration was nonlinear, as illustrated in curve B in Fig. 5. Curve A is the steady state rate of the enzyme initiated reaction. If the data of line B are plotted versus the square of the enzyme concentration, a linear increase in activity is observed. In data not shown, when the enzyme reaction was initiated with MgATP, the activity obtained by taking the slope of the initial fast phase of the burst transient also increased in a nonlinear fashion with the amount of enzyme used, similar to that shown in Fig. 5. Effect of substrate concentration. When the reaction was initiated with enzyme, the pseudo-first order rate constant did not show any apparent correlation with changes in MgATP (0.1-0.8 111~) and HCOs- (2-20 111~)concentrations (data not shown). The R,,f also did not show dependence on MgATP and HC03- concentrations. However, when pyruvate concentrations were varied and the reaction initiated with enzyme, K’ decreased with increasing pyruvate concentrations up to 1 mu and then remained constant up to 5 mu (Fig. 6). The R,,f value declined in a similar but more gradual manner. Similar results were I
I
i
0.14 t
70 0.10 f -20. ; -1 0.0 ENZYME
1~1 addition)
FIG. 4. Effect of enzyme concentration on the activ-
ity and the lag phase. (A) (0) Activities were determined from the slopes of the final steady state phase of the progress curves with different amounts of enzyme. Enzyme was added last to initiate the reaction. Other assay conditions are described under Materials and Methods. (B) (0) Pseudo-first order rate constant, k’, versus amount of enzyme. k’ were determined as described by Hatfield et al. (9).
662
YIP AND
PI
ENZYME
FIG. 5. Effect of protein concentration on activity under different assay conditions. Curve (A) (0): The enzyme was added last to initiate the reaction and the velocity was obtained from the linear steady state portion of the progress curve. Curve (B) (0): The enzyme was incubated in a cuvette with buffer, acetylCoA, NADH and malate dehydrogenase for 2 min and MgATP, HC03-, pyruvate were added (70 dul) at the same time to initiate the reaction and the velocity was obtained from the initial linear portion of the reaction progress curve. Preincubation times of 0.5 to 10 min have no effect on the initial velocity. Other conditions of assay are as described in Materials and Methods.
obtained when pyruvate was used to initiate the reaction. Double reciprocal plots obtained with varying MgATP and HC03 concentrations were linear while the double reciprocal plot obtained with varying pyruvate was biphasic, with a transition at a concentration of about 0.4 mu pyruvate, similar to previous observations with the chicken liver (lo), rat liver (ll), and sheep kidney enzymes (12). The K, values for the substrates for the beef liver enzyme were: 0.04 mru for MgATP; 3.3 mM for HCOs-; and 0.083 mu and 0.33 mu for the low’and high concentrations of pyruvate. These values are comparable to those obtained for rat liver (11) and chicken liver enzyme (10). Effect of temperature. When the temperature was lowered from 30°C to lO”C, the lag for the enzyme and pyruvate initiated reactions and the burst for ATP initiated reactions were still observed. As shown in Fig. 7, when the reaction was initiated with addition of enzyme, the initial rate and the steady state rates of the reaction progress curves are linear in an Arrhenius plot in the
RUDOLPH
temperature range of 5-30°C. Assuming that the levels of the substrates used were saturating at all the temperatures tested, the energy of activation for the pyruvate carboxylase reaction calculated for both cases was 8.3 kcal, which is in good agreement with the value found previously of 8.4 kcal for rat liver enzyme (3) and 8.1 kcal for chicken liver enzyme (13). When the pseudo-first order rate constant for the conversion from one form of the enzyme to another was analyzed, an apparently linear relationship was obtained (Fig. 7), indicating that the transition may be interpreted as a simple chemical process. The activation energy of the process was calculated to be. 1.7 kcal. Effect of salt and PH. Experiments were performed to determine if changes in pH and ionic strength affect the lag. As shown in Table II, the lag phenomenon was still observed at pH 7.8 and added 0.1 M KC1 when the reaction was initiated with enzyme. However, the difference between the initial velocity and final velocity decreased (i.e. R,,f increased). Similar behavior was
6
.
0.6 RI/f
0.4 0.2 0
I
2 PYRUVATE
4
3
5
, mM ,
FIG. 6. Effect of pyruvate concentration on the lag phase. Progress curves obtained at several pyruvate concentrations were treated according to the graphical procedures of Hatfield et al. (9) for analyzing a lag phase involving a first order process. (A) Pseudo-first order rate constants (k’) and (B) the ratio of the initial slow rate to the steady state rate were plotted versus pyruvate concentration. Assay conditions were as described under Materials and Methods and a constant amount of enzyme was added last to initiate the reaction. Reaction velocity (measured from the fast phase) at 5 mM pyruvate is 0.027 AOD&20 s.
BOVINE
LIVER (I/T)
PYRUVATE x IO’K
CARBOXYLASE
TRANSIENTS (I/T)
x IO3
663
K
FIG. 7. Effect of temperature on the kinetic parameters of pyruvate carboxylase. (A) The logarithm of the maximal velocity (AA&40 s) for the forward reaction is plotted against the reciprocal temperature (“K). (O), the velocity measured from the tangent of the initial slope of the lag; (A), the velocity measured from the steady state linear phase of the reaction progress curve. (B) The log of the pseudo-first order rate constant, obtained from the analysis of the lag by the procedures of Hatfield et al. (9), plotted against the reciprocal temperature in “K. Conditions of the assay, except the temperature, were as described under Materials an> Methods. Amount of enzyme used was constant and the activity at 30°C was 0.05 A.&/40 s.
is linear with time (3, 14-16). However, there were several observations that indicated that the enzyme is hysteretic. In studIonic strength, pH” Initiated with R,,f ies on Mg2+ activation of sheep kidney enNo salt, pH 7.0 Enzyme 0.55 zyme, Bais and Keech (17) have shown that 0.1 M KCl, pH 7.0 Enzyme 0.74 there is a lag phse in the activation by No salt, pH 7.8 Enzyme 0.78 Mp. A lag was also observed when the rat 0.1 M KCl, pH 7.8 Enzyme 0.85 liver enzyme was incubated with salt (6). No salt, pH 7.0 Pyruvate 0.8 0.1 M KCl, pH 7.0 Pyruvate 0.85 Recently, in a study on the binding of aceNo salt, pH 7.8 Pyruvate 0.95 tyl-CoA to chicken liver enzyme, Frey and 0.1 M KCl, pH 7.8 Pyruvate 1.00 Utter (18) have reported that a lag in the a Other assay conditions, except as indicated, were reaction rate was observed even when the the same as described under Materials and Methods. enzyme was preincubated with acetyl-CoA. At pH 7.0, with no added KCl, enzyme activity was Previous assays in this enzyme were gen0.001 A OD 340“,J.s. The activity at pH 7.8 was about erally performed at pH 8 and/or 0.1 M KC1 twice that at pH 7.0. The R,,, is the average of at least (3, 14-16). The studies carried out here four determinations. were at a more physiological pH and no additional salt. It should be noted that sufobserved when the reaction was initiated ficient K+ (0.05 M) was present to activate with pyruvate. In fact, at pH 7.8 and added the enzyme and little difference was seen in 0.1 M KCl, no pyruvate induced lag was total activity between 0.05 and 0.15 M K+. observed. In the presence of added 0.1 M Presence of the added salt in the assay KCl, the reaction progress curves generated causes a decrease in the transient effect, as by adding MgATP last were linear for at is illustrated by the data of Table II. Thus, least 1 min and the burst transient was not at pH 8 the transient is not as apparent, apparent at either pH 7 and 8. and could be overlooked by the investigator particularly when using coupled enzyme asDISCUSSION say system. Earlier studies on chicken liver, rat liver, The use of the independent assay methand sheep kidney pyruvate carboxylase ods demonstrate that the transient obhave generally shown that the reaction rate served is not an artifact of the coupled TABLE
II
EFFECT OF SALT AND pH ON LAG TRANSIENT
664
YIP AND RUDOLPH
enzyme assay system. Recently, Easterbrook-Smith et al. (19) showed that enzyme bound CO2 was not stoichiometrically transferred to pyruvate. Therefore, it could be possible that the lag was caused by this nonequivalent transfer because both the coupling enzyme assay and [‘4C]CO~ incorporation assay measured oxalacetate production. However, the lag was still observed with [Y-~~P]ATP assay, ruling out the possibility that the nonequivalent transfer was the cause of the lag. There appear to be two transient effects, one induced by substrates and the other induced by acetyl-CoA. The transient effects can be interpreted as a slow transition between two forms of the enzyme with different catalytic activity. The transition may be due to a change in conformation or in the quaternary structure of the protein or both. The effects of MgATP and pyruvate are antagonistic to each other. MgATP is able to induce the enzyme to shift from a more active conformation to a less active one; pyruvate and bicarbonate cause the enzyme to shift from a less active to a more active conformation. The effect of the nucleotide appears to be predominant over the effect of pyruvate at lower MgATP and pyruvate concentrations. The transient induced by acetyl-CoA was studied in more detail. The observations that the transition was pseudo-first order with respect to time at different enzyme concentration (Fig. 4) and the Arrhenius plot was linear with a low energy of activation (Fig. 7) are all consistent with a conformational change. Even though a conformational change is strongly implicated as the mechanism for the slow transient, under conditions where the slow transient was abolished, e.g., when the enzyme was preincubated with acetylCoA in the cuvette and the reaction was initiated by adding all the substrates together, the non-linear dependence of activity versus enzyme concentration (Fig. 5) strongly suggests that an association process takes place. It is not understood what the interaction between conformational change and change in quaternary structure is, and how it will affect the transient and the catalytic rate of the reaction. Taylor et al. (20) have shown that at low enzyme and
salt concentrations similar to those of this study, the enzyme is a tetramer in the presence of acetyl-CoA but a dimer in its absence. Preincubation of the beef liver enzyme abolished the lag. This result differs from that reported on the chicken liver enzyme, in which case the lag was not abolished (18). The difference between the two results may be due to the species difference between the enzymes. The chicken liver enzyme has been shown to differ from the rat liver and beef liver enzyme in immunological properties and catalytic properties (1,
4). In previous reports on rat liver, chicken liver and sheep kidney enzymes and also in the present report on beef liver, the double reciprocal plot with pyruvate as the varied substrate was biphasic. Recently, Easterbrook-Smith et al. (19) presented an interesting study to explain the biphasic plot. They found the biotin-bound carbon dioxide was not stoichiometrically transferred to pyruvate at low pyruvate concentration and the transfer was stoichiometric at higher pyruvate concentration. The results presented in this report on the effect of pyruvate concentration on the conformation of the enzyme are interesting relative to that finding. As shown in Fig. 6, at low pyruvate concentration (less than 1 mu) and saturating MgATP and HC03-, the enzyme did not exhibit a lag (R,p = 1) while at high pyruvate concentration, the initial rate was slower than the final steady rate (Rslf = 0.7). The rate of the transient also exhibited changes around 1 mu pyruvate. Since both R,,f and k’ varied with pyruvate concentration, it is tempting to speculate that the stoichiometry of carbon dioxide transfer to pyruvate may depend on the conformational state of the enzyme, which in turn depends on the pyruvate concentration. At low pyruvate concentrations, the enzyme remains mainly in the less active conformation, which is not able to transfer carbon dioxide efficiently. At high pyruvate concentrations, the enzyme is shifted to a more active conformation, and nearly stoichiometric transfer of carbon dioxide proceeds. Regulatory implications: The transient
BOVINE
LIVER
PYRUVATE
effect reported here has not been shown to take place in uivo. However, there are examples that an enzyme exhibiting transient properties in vitro also exhibited these properties in vivo (e.g. yeast hexokinase (21)). Thus, the hysteretic properties of the enzyme may be a part of its regulatory mechanisms. In general, two factors are considered to be the primary mechanism for regulation of this enzyme (18, 22). These are acetyl-CoA activation (governing the amount of active enzyme) and the amount of available pyruvate. The introduction of the concept of hysteretic enzyme clearly adds another dimension to the metabolic regulation of the enzyme. According to Frieden (5), a hysteretic enzyme can function as a time-dependent buffer while at the same time slowly responding to changes which will eventually alter the kinetic characteristics of the enzyme to correspond to the altered level of metabolites in the cell. The buffering capacity of such an enzyme system, which depends upon the rate of conversion of one form of the enzyme to another, serves as a mechanism to prevent immediate changes in the concentration of other metabolites in the particular pathway, thus allowing the rate of other intersecting pathways which use a common starting metabolite to be maintained. Both pyruvate and oxalacetate participate in a complexity of metabolic pathways, and it is logical that their levels are tightly regulated in this manner. It is interesting that other enzymes which act on pyruvate are known to exhibit a lag transient. These include phosphoenolpyruvate carboxylase (listed in (23)) and pyruvate kinase (B), suggesting the importance of this mode of control for pyruvate metabolism in vivo. It is also interesting to point out that, to the authors’ knowledge, this is the first demonstration that an enzyme exhibits both a burst and a lag transient depending on the conditions of the assay. ACKNOWLEDGMENTS The authors wish to thank Dr. Scott Power for assistance in the use of the Interdata computer which
CARBOXYLASE
TRANSIENTS
was kindly made available mer.
665
by Professor Graham Pal-
REFERENCES
4. 5. 6.
7. 8. 9. 10. 11.
UTTER, M. F., AND SCRUTTON, M. C. (1969) Curr. Top. Cell. Regul. 1,253-296. LING, A. M., AND KEECH, D. B. (1966) Enzymologia 30,367-380. MCCLURE, W. R., LARDY, H. A., AND KNEIFEL, H. P. (1971) J. Biol. Chem. 246,3569-3578. UTTER, M. F., BARDEN, R. E., AND TAYLOR, B. L. (1974) Adv. Enzymol. 40, l-72. FRIEDEN, C. (1970) J. Biol. Chem. 245,5788-5799. NAICASHIMA, K., RUDOLPH, F. B., WAKABAYASHI, T., AND LARDY, H. (1975) J. Biol. Chem. 250, 331-336. MCCLURE, W. R., LARDY, H. A., AND CLELAND, W. W. (1971) J. Biol. Chem. 246,3584-3590. BADWEY, J. A., AND WESTHEAD, E. W. (1976) J. Biol. Chem. 251,5600-5606. HATFIELD, G. W., RAY, W. J., JR., AND UMBARGER, H. E. (1970) J. Biol. Chem. 245,1748-1754. SCRUTTON, M. C., KEECH, D. B., AND UTTER, M. F. (1965) J. Biol. Chem. 240.574-581. MCCLURE, W. R., LARDY, H. A., WAGNER, M., AND CLELAND, W. W. (1971) J. Biol. Chem. 246,
3579-3583. 12. TAYLOR, H., NIELSEN, J., AND KEECH, D. B. (1969) B&hem. Biophys. Res. Commun. 37, 723-728. 13. KEECH, D. B., AND UTTER, M. F. (1963) J. Biol.
Chem. 238,2609-2614. 14. UTTER, M. F., AND KEECH, D. B. (1963) J. Biol.
Chem. 238,2603-2608. 15. WIMHURST, J. M., AND MANCHESTER, K. L. (1970) Biochem. J. 120, 79-93. 16. SEUFERT, D., HERLEMANN, E. M., ALBRECHT, E., AND SEUBERT, W. (1971) Hoppe-Seyler’s Z.
Physiol. Chem. 352,459-478. 17. BAIS, R., AND KEECH, D. B. (1972) J. Biol. Chem. 241,3255-3261. 18. FREY, W. H., III, AND UITER, M. F. (1977) J. Biol.
Chem. 252,51-56. 19. EASTERBROOK-SMITH, S. B., HUDSON, P. J., Goss, N. H., KEECH, D. B., AND WALLACE, J. C. (1976) Arch. Biochem. Biophys. 176.709-720. 20. TAYLOR, B. L., FREY, W. H., BARDEN, R. E., SCRUTTON, M. C., AND UTTER, M. F. (1978) J.
Biol. Chem. 253,3062-3069. 21. REITZER, L. J., AND NEET, K. E. (1974) Biochem. Biophys. Acta 341,201-212. 22. MCCLURE, W. R., AND LARDY, H. A. (1971) J.
Biol. Chem. 246,3591-3596. 23. AINSLIE, G. R., JR., SHILL, J. P., AND NEET, K. E. (1972) J. Biol. Chem. 247, 7088-7096.
Hysteretic
Kinetic Behavior of Beef Liver Pyruvate Carboxylase’ BERWIN
P. YIP2 AND FREDERICK
B. RUDOLPH
Department of Biochemistry, Rice University, Houston, Texas 77001 Received February
22, 1978; revised July 27, 1978
Beef liver pyruvate carboxylase can exhibit transients of enzyme activity depending on prior incubation conditions. At pH 7.0, when the reaction is initiated with MgATP (after incubation with the other reaction components) an initial burst of activity occurs which decays to a slower rate. If the reaction is initiated by addition of enzyme, acetyl-CoA, or pyruvate, a lag occurs followed by an increase in activity to a steady state level after about one to two minutes. The lag transient has been analyzed by a pseudo fast order rate equation derived by Hatfield et al. (J. Biol Chem. 245, 1748-1754, 1970) assuming a slow transition from one enzyme conformation to another. The rate constant for the transition was independent of enzyme concentration and MgATP and bicarbonate concentrations in the range tested, but was markedly dependent on pyruvate concentration. From the temperature dependence of the rate of transition, a value of 1.7 kcal was calculated for the conformational change. At pH 8 with additional salt (0.1 M KCl), the transient effect was diminished but can still be observed in some cases. It is suggested that biphasic double reciprocal plots observed with pyruvate may be due to a pyruvate-induced conformational change to a more active form at high pyruvate levels.
Pyruvate carboxylase (pyruvate:COz ligase (ADP forming), EC 6.4.1.1) catalyzes the reaction:
two classes of vertebrate pyruvate carboxylase. The enzymes isolated from chicken liver, turkey liver (l), and sheep kidney (2) are similar as are the ones from rat (3) and calf liver (1). During studies on the beef liver pyruvate carboxylase, a lag in the assay was repeatedly observed when the reaction was initiated by the addition of the enzyme. A few previous investigations with pyruvate carboxylase from various sources have mentioned such phenomena (4), but no studies were carried out to characterize it. This report describes the results of a detailed investigation into this effect. The possible metabolic significance of this “hysteretic” (5) behavior is discussed.
Pyruvate
+ MgATP2acetyl-CoA + HCOS- \ ’ oxalacetate Mg2+, K+ + MgADP- + Pi.
This reaction serves a gluconeogenic role in liver, kidney, and some microorganisms and an anaplerotic role in other tissues and species. It has been suggested that many factors are involved in its regulation (1). The enzyme has been studied from various sources and is a tetrameric protein containing bound metals and needing several activators (1). By the criteria of sensitivity to cold inactivation, immunochemical studies, and relationship between the quaternary structure and activity, there appear to be
MATERIALS
AND
METHODS
Beef liver pyruvate carboxylase was purified using a modification of the procedure developed for the rat liver enzyme (3). The lyophilized mitochondrial powder was extracted with 0.05 M KPi buffer, pH 7.0, containing 5 mM ATP, 5 mM MgS04 and 0.5 mu EDTA, followed by a 28-35% saturation of ammonium sulfate precipitation. The heat step described in (3) was omitted. The precipitate was dissolved in 0.1 M Tris-Cl buffer at pH 7.2 containing 0.1 mM EDTA, 1
’ This research was supported in part by Grant C582 from the Robert A. Welch Foundation and Grant CA 14030 from the National Cancer Institute, DHEW. ’ Recipient of a Robert A. Welch Foundation Predoctoral Fellowship. Present address: Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin. 657
0003-9861/78/1912-0657$02.00/O Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.
658
YIP AND
mu dithiothreitol and 12.6 g/100 ml (NH&S04. After centrifugation to separate the precipitate, solid ammonium sulfate was added to the supernatant to a concentration of 50 g/100 ml and the precipitate obtained was extracted successively as described in (3). The 14.4 and 12.6 g of ammonium sulfate/100 ml fractions were pooled and the protein was precipitated by adding solid ammonium sulfate to the pooled fractions to a final concentration of 50 g/100 ml. The precipitate was dissolved in a minimal volume of 0.1 M Tris-Cl, 1 mu EDTA, 1 mM dithiothreitol, pH 7.2 and applied to a Sepharose 6B column (2.4 x 25 cm) previously equilibrated with the same buffer. The fractions containing the enzyme activity were pooled and concentrated by ammonium sulfate precipitation. The specific activity at pH 8.0 was 6-10 units/mg protein. Attempts were also made to purify the enzyme further by DEAE column chromatography as described in (6) and on sucrose gradients (lo-258 linear gradient). The specific activity increased somewhat but the protein obtained was not homogeneous based on polyacrylamide gel electrophoresis. The enzyme obtained from the Sepharose column did not show any contaminating enzyme activity which would interfere in the enzyme assays, and was routinely used in most experiments. In addition, enzyme processed through additional DEAE chromatography and sucrose gradient purification steps exhibited the same transients as the enzyme from the Sepharose column. The concentrated enzyme, which was stored in sucrose buffer (3), was dialyzed overnight against 0.1 M Tris-Cl, 0.1 mru EDTA and 1 mM dithiothreitol, pH 7.2, before use. The materials were obtained from the following indicated sources: acetyl-CoA, dithiothreitol, NADH, Sigma; ATP, Calbiochem; pyruvic acid, J. T. Baker; Na[‘%]HC03, Amersham/Searle; [Y-~‘P]ATP, tetra(triethylammonium) salt, New England Nuclear; Sepharose 6B, Pharmacia. Assays. The enzyme was routinely assayed by the malate dehydrogenase coupling enzyme assay as described previously (3). pH was maintained at pH 7.0 in 0.1 M Hepes3 buffer. The reactions were monitored in a thermostated Cary 118 spectrophotometer. The concentrations (mM) of the assay mixture, in a total volume of 1 ml, were: pyruvate, 5; KHCOJ, 20; MgATP, 0.8; acetyl-CoA, 0.16; NADH, 0.16. The components were incubated at 30°C prior to initiation of the reaction. The incorporation of radioactive carbon dioxide into oxalacetate was assayed as described in (7) with the following modifications. The reaction mixture contained, in a total volume of 0.25 ml, the following mu concentrations of substrates: Hepes, 100, pH 7.0; pyruvate, 5; KHCO,; 20, MgATP, 2.4; acetyl-CoA, 0.16; ’ Abbreviations used: Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; TCA, trichloroacetic acid.
RUDOLPH [“C]HC03-, 3.3 (sp. act. 60.2 mCi/mmol). A lo-p1 sample of the reaction was transferred at timed intervals (usually 10 s for fit 10 samples and 20 s for next 10 samples) into 50 )d of 3.6% cold TCA in a small disposable centrifuge tube, followed by thorough mixing. The samples were neutralized with KOH. Malate dehydrogenase and NADH (final concentration, 0.3 mM) were then added to the mixture to convert the oxalacetate to malate. After 10 min at room temperature, 50 al of 10% cold TCA was added, and the mixture was centrifuged (1006 rpm). Residual radioactive carbon dioxide was driven off by bubbling with carbon dioxide for 10 min. Aliquots (100 ~1) were removed for determination of [‘%]malate formed. They were counted in a liquid scintillation spectrophotometer using a scintillation fluid with the following composition (toluene, 2100 ml; ethanol, 1230 ml, 2,5-diphenyloxazole, 12 g; 1,4-bis[2-(5phenyloxazolyl)]benzene, 0.3 g). The reaction was also followed by determination of the “Pi released from [y-32P]ATP. The assay mixture contained in a volume of 0.25 ml, the following mM concentrations: pyruvate, 5; acetyl-CoA, 0.16; KHCO$, 20; MgATP, 0.16; Hepes, 100, pH 7.0 and [y-3ZP]ATP, 8.9 X lOma (sp. act. 28.2 Ci/mmol). A lo-$ aliquot of the reaction mix was transferred at timed intervals into 50 al of 6% cold TCA in a small centrifuge tube. One hundred microliters of isobutanol; benzene (Hz0 saturated) (1:l) was added and the solution vortexed for 15 s. Ten microliters of a 40 mM molybdate solution (in 4 N H&04) was then added to each of the centrifuge tubes and the mixture was vortexed vigorously for 30 s. The samples were then centrifuged for 2 mm (1006 rpm). Aliquots of the organic layer (50 )rl) were removed and counted in the medium described above. RESULTS
During routine assays in studies with beef liver pyruvate carboxylase, it was observed that, under some conditions, the reaction velocity changed with time. When the reaction was initiated with the enzyme or pyruvate, the reaction progress curve exhibited a lag, i.e., the reaction rate was slow at the beginning and increased over a period of a minute to a steady state rate. Bicarbonate initiated reactions also exhibited a lag period, but with a shorter time period prior to steady state attainment. A different effect was observed when the reaction was initiated with MgATP. The initial rate of the reaction exhibited a burst transient with the reaction starting at a fast rate and decaying slowly. In general, a steady state reaction was not observed with the MgATP initiated reaction. The coupled enzyme assay was used in
BOVINE
LIVER
PYRUVATE
‘i
FIG. 1. Radioactive assays of pyruvate carboxylase. Details are described in Materials and Methods. (0) [‘%]HC03incorporation; ATP initiated reaction. (0) [‘%]HC03incorporation; enzyme initiated reaction. (x) [32P]phosphate release; enzyme initiated reaction (a different amount of activity was used for this assay than for the other two).
initial studies and the lag effect could be attributed to the inherent lag in the coupling enzyme system. However, malate dehydrogenase was always present in excess, and additions of more coupling enzyme did not affect the observed lag. Independent methods of assay were used to confirm the validity of the assay, where the enzyme reaction was followed by measuring the amount of [‘4C]bicarbonate incorporated into oxalacetate and by the release of radioactive inorganic phosphate from [T-~‘P] ATP as shown in Fig. 1. The lag was also present when the reaction was initiated by addition of the enzyme and distinctly different from the transient observed when the reaction was initiated with MgATP. To quantitate the extent of the lag phase, the ratio (R,,f) of the initial reaction velocity (initial slow phase) to the steady state velocity (final fast velocity) was calculated (8). Initial velocity measurements were made about 18 sec. after the addition of the component to initiate the reaction, which was the time required for mixing and instrument set-up and thus do not measure the actual initial velocity. However, the R,,f values still provide a good estimate of the extent of the lag phase as measurements were made in a consistent manner. Under
CARBOXYLASE
659
TRANSIENTS
the standard usual conditions, when the reaction was initiated with the enzyme, the Rslfvaried within the range of 0.5 to 0.8 for different preparations of the enzyme. To evaluate the lag effect, a series of incubations with different combinations of substrates was done (Table I). The first three conditions are as described above. A MgATP initiated reaction is not listed because a steady state rate was not usually observed. However, by using the steady state rate obtained from the enzyme initiated reaction as the steady state rate for the MgATP initiated reaction, the R,,f is 1.5. Incubations 2, 3, and 5 illustrate that a lag is observed only when the enzyme is preincubated with MgATP. Enzyme preinTABLE
I
EFFECT OF INCUBATION WITH SUBSTRATES ON LAG PHASE Reaction 1. 2. 3. 4. 5. 6. 7. 8.
9. 10.d 11.’
initiated
with”
Enzyme Pyruvate HCOaATP + pyruvate HC03- + pyruvate ATP + HCOZEnzyme preincubated with dithiothreitol Enzyme preincubated dithiothreitol with and bovine serum albumin ATP + pyruvate + HCO,ATP + pyruvate + HCOaATP + pyruvate + HCOa-
K
V
0.036 0.050 0.051 0.104 0.047 0.080 0.038
0.066 0.067 0.065 0.104 0.072 0.080 0.070
0.54 0.75 0.78 1.00 0.66 1.00 0.54
0.048
0.086
0.56
0.062
0.062
1.90
0.081
0.081
1.00
0.092
0.092
1.06
k/f
a Assay was as described under Materials and Methods. Final enzyme concentration was the same for ah cases. The enzyme was incubated with ah the components except those to be added as indicated for 2 min at 30°C and the reaction was initiated with the component indicated. The numbers represent the average of at least four determinations. * V,, initial velocity in A OD340.,/40 s, was taken at the earliest time possible (usually after about 20 s of addition of enzyme or substrate). ’ V,, final steady state velocity in A ODsO .,/40 s, was measured 2 min after the start of the reaction, and was linear for at least 1 min. d Dithiothreitol was also present in the cuvette. ‘Dithiothreitol and bovine serum albumin were also present in the cuvette.
660
YIP AND
cubated with dithiothreitol(O.2 mu) or bovine serum albumin (0.5 mg/ml) also exhibited the lag transient. When the enzyme was added into the cuvette first (with buffer and acetyl-CoA) and the reaction initiated with all the substrates (70-g addition), no transient was observed and the reaction progress was linear with time for more than 2 min. Inclusion of dithiothreitol and bovine serum albumin did not have any effect on the linearity of the reaction progress, although inclusion. of these two reagents activated the enzyme slightly. Beef liver pyruvate carboxylase is functionally dependent on acetyl-CoA for activity. When acetyl-CoA is omitted from the assay mixture, and the enzyme is incubated with all the substrates and the coupling enzyme system, no reaction was detected. Addition of acetyl-CoA restores the activity and the reaction exhibited a lag phase (Fig.
RUDOLPH
between two enzyme species of different intrinsic activity. The transition can be analyzed according to the equation outlined by Hatfield et al. (9). A similar equation has also been described by Frieden (5). As illustrated in the inset of Fig. 2, graphical analysis of the data by this method yielded apparently linear first order log plots, suggesting that the analysis is descriptive of the phenomena observed. The pseudo-first order constant for the conversion of the less active enzyme species to the more active form, k’, can be obtained from the slope of the log plot (9). In this analysis, a knowledge of the true initial velocity was not
0.'
2).
The above studies were carried out at saturating levels of substrates. When the experiments were done with Km levels of substrates (i.e., 0.08 mu MgATP, 2 mu bicarbonate, and 0.5 mu pyruvate), a somewhat different behavior of the enzyme was observed. No lag was observed with pyruvate added last to initiate the reaction but a lag was still observed when the enzyme was used to initiate the reaction. When the reaction was studied by initiating with all three substrates, the reaction trace showed a burst transient, gradually slowing down. The burst was also observed when MgATP was used to initiate the reaction. This phenomenon was apparently not caused by substrate depletion at the lower substrate concentrations used, because the reaction progress initiated by pyruvate or enzyme still showed a linear rate when the same amount of reaction had taken place. The transient was dependent on substrate concentrations. Thus, in Fig. 3, when the MgATP concentration is markedly increased (arrow), during the steady state of a reaction in the presence of non-saturating MgATP, the reaction progress goes through another transient. Similar results were obtained with pyruvate as the varying substrate (data not shown). The transient indicates a slow transition
0.3
‘+%a
0.2
0.1
I
0
20
40 TIME
60
SO
lrccl
PIG. 2. Reaction progress curve generated by adding acetyl-CoA to initiate the reaction. The enzyme was incubated with all the reaction components minus acetyl-CoA. No reaction was detected. Acetyl-CoA was then added (10 $ addition; final concentration 0.13 mg/ml) to activate the enzyme. Inset: Analysis of lag phase involving a first order process as described by Hatfield et al. (9). A A, represents the maximum difference in absorbance change between an imaginary linear progress curve with the reaction velocity the sane as the steady state velocity of the transient and the progress curve exhibiting the lag. A At represents the difference between the two progress curves at time t of the lag phase. See Ref. (9) for details of the rate equation and a graphical illustration of the analysis. The pseudo-Srst order rate constant for the line shown
is 0.05 s-1.
BOVINE LIVER PYRUVATB 0.3
0.2 AA 3.0
0.1
0
50
100 TIME
150 (set
200
)
FIG. 3. Reaction progress curve with steady state addition of supplemental substrate. Curve A is obtained with 0.04 mre MgATP, the other substrate concentrations as described in Materials and Methods. At the srrow, MgATP was added to yield a final concentration of 0.34 mru (curve B). Curve C is that obtained with 034 mru MgATP, with MgATP added last. Curve D is that obtained with 0.34 mru MgATP with enzyme added last.
required. Subsequent calculations of the rate of transition were determined by linear regression using a digital computer. Since it was not possible to obtain steady state rates for MgATP initiated reaction, further studies were carried out only with enzyme or pyruvate initiated reactions. Saturating substrate concentrations were employed because one of the conditions for the equation derived by Hatfield et al. (9) was that the substrate concentration remained constant throughout the time course of the analysis. Effect of enzyme concentration. Progress curves generated by adding different amounts of the same enzyme preparation to initiate the reaction were analyzed and the pseudo-first order rate constants obtained were plotted versus the amount of the enzyme used (Fig. 4). The pseudo-first order rate constants stayed fairly constant over the enzyme concentrations used. The R,,f values obtained were also constant (0.65 f 0.05). Also, as shown in Fig. 4, the velocity obtained by taking the slope of the steady state portion of the progress curve was proportional to the concentration of
CARBOXYLASE
661
TRANSIENTS
the enzyme added. When the reaction was initiated with all three substrates added together after preincubation with acetylCoA, the reaction progress curves were linear, but the plot of the velocity versus enzyme concentration was nonlinear, as illustrated in curve B in Fig. 5. Curve A is the steady state rate of the enzyme initiated reaction. If the data of line B are plotted versus the square of the enzyme concentration, a linear increase in activity is observed. In data not shown, when the enzyme reaction was initiated with MgATP, the activity obtained by taking the slope of the initial fast phase of the burst transient also increased in a nonlinear fashion with the amount of enzyme used, similar to that shown in Fig. 5. Effect of substrate concentration. When the reaction was initiated with enzyme, the pseudo-first order rate constant did not show any apparent correlation with changes in MgATP (0.1-0.8 111~) and HCOs- (2-20 111~)concentrations (data not shown). The R,,f also did not show dependence on MgATP and HC03- concentrations. However, when pyruvate concentrations were varied and the reaction initiated with enzyme, K’ decreased with increasing pyruvate concentrations up to 1 mu and then remained constant up to 5 mu (Fig. 6). The R,,f value declined in a similar but more gradual manner. Similar results were I
I
i
0.14 t
70 0.10 f -20. ; -1 0.0 ENZYME
1~1 addition)
FIG. 4. Effect of enzyme concentration on the activ-
ity and the lag phase. (A) (0) Activities were determined from the slopes of the final steady state phase of the progress curves with different amounts of enzyme. Enzyme was added last to initiate the reaction. Other assay conditions are described under Materials and Methods. (B) (0) Pseudo-first order rate constant, k’, versus amount of enzyme. k’ were determined as described by Hatfield et al. (9).
662
YIP AND
PI
ENZYME
FIG. 5. Effect of protein concentration on activity under different assay conditions. Curve (A) (0): The enzyme was added last to initiate the reaction and the velocity was obtained from the linear steady state portion of the progress curve. Curve (B) (0): The enzyme was incubated in a cuvette with buffer, acetylCoA, NADH and malate dehydrogenase for 2 min and MgATP, HC03-, pyruvate were added (70 dul) at the same time to initiate the reaction and the velocity was obtained from the initial linear portion of the reaction progress curve. Preincubation times of 0.5 to 10 min have no effect on the initial velocity. Other conditions of assay are as described in Materials and Methods.
obtained when pyruvate was used to initiate the reaction. Double reciprocal plots obtained with varying MgATP and HC03 concentrations were linear while the double reciprocal plot obtained with varying pyruvate was biphasic, with a transition at a concentration of about 0.4 mu pyruvate, similar to previous observations with the chicken liver (lo), rat liver (ll), and sheep kidney enzymes (12). The K, values for the substrates for the beef liver enzyme were: 0.04 mru for MgATP; 3.3 mM for HCOs-; and 0.083 mu and 0.33 mu for the low’and high concentrations of pyruvate. These values are comparable to those obtained for rat liver (11) and chicken liver enzyme (10). Effect of temperature. When the temperature was lowered from 30°C to lO”C, the lag for the enzyme and pyruvate initiated reactions and the burst for ATP initiated reactions were still observed. As shown in Fig. 7, when the reaction was initiated with addition of enzyme, the initial rate and the steady state rates of the reaction progress curves are linear in an Arrhenius plot in the
RUDOLPH
temperature range of 5-30°C. Assuming that the levels of the substrates used were saturating at all the temperatures tested, the energy of activation for the pyruvate carboxylase reaction calculated for both cases was 8.3 kcal, which is in good agreement with the value found previously of 8.4 kcal for rat liver enzyme (3) and 8.1 kcal for chicken liver enzyme (13). When the pseudo-first order rate constant for the conversion from one form of the enzyme to another was analyzed, an apparently linear relationship was obtained (Fig. 7), indicating that the transition may be interpreted as a simple chemical process. The activation energy of the process was calculated to be. 1.7 kcal. Effect of salt and PH. Experiments were performed to determine if changes in pH and ionic strength affect the lag. As shown in Table II, the lag phenomenon was still observed at pH 7.8 and added 0.1 M KC1 when the reaction was initiated with enzyme. However, the difference between the initial velocity and final velocity decreased (i.e. R,,f increased). Similar behavior was
6
.
0.6 RI/f
0.4 0.2 0
I
2 PYRUVATE
4
3
5
, mM ,
FIG. 6. Effect of pyruvate concentration on the lag phase. Progress curves obtained at several pyruvate concentrations were treated according to the graphical procedures of Hatfield et al. (9) for analyzing a lag phase involving a first order process. (A) Pseudo-first order rate constants (k’) and (B) the ratio of the initial slow rate to the steady state rate were plotted versus pyruvate concentration. Assay conditions were as described under Materials and Methods and a constant amount of enzyme was added last to initiate the reaction. Reaction velocity (measured from the fast phase) at 5 mM pyruvate is 0.027 AOD&20 s.
BOVINE
LIVER (I/T)
PYRUVATE x IO’K
CARBOXYLASE
TRANSIENTS (I/T)
x IO3
663
K
FIG. 7. Effect of temperature on the kinetic parameters of pyruvate carboxylase. (A) The logarithm of the maximal velocity (AA&40 s) for the forward reaction is plotted against the reciprocal temperature (“K). (O), the velocity measured from the tangent of the initial slope of the lag; (A), the velocity measured from the steady state linear phase of the reaction progress curve. (B) The log of the pseudo-first order rate constant, obtained from the analysis of the lag by the procedures of Hatfield et al. (9), plotted against the reciprocal temperature in “K. Conditions of the assay, except the temperature, were as described under Materials an> Methods. Amount of enzyme used was constant and the activity at 30°C was 0.05 A.&/40 s.
is linear with time (3, 14-16). However, there were several observations that indicated that the enzyme is hysteretic. In studIonic strength, pH” Initiated with R,,f ies on Mg2+ activation of sheep kidney enNo salt, pH 7.0 Enzyme 0.55 zyme, Bais and Keech (17) have shown that 0.1 M KCl, pH 7.0 Enzyme 0.74 there is a lag phse in the activation by No salt, pH 7.8 Enzyme 0.78 Mp. A lag was also observed when the rat 0.1 M KCl, pH 7.8 Enzyme 0.85 liver enzyme was incubated with salt (6). No salt, pH 7.0 Pyruvate 0.8 0.1 M KCl, pH 7.0 Pyruvate 0.85 Recently, in a study on the binding of aceNo salt, pH 7.8 Pyruvate 0.95 tyl-CoA to chicken liver enzyme, Frey and 0.1 M KCl, pH 7.8 Pyruvate 1.00 Utter (18) have reported that a lag in the a Other assay conditions, except as indicated, were reaction rate was observed even when the the same as described under Materials and Methods. enzyme was preincubated with acetyl-CoA. At pH 7.0, with no added KCl, enzyme activity was Previous assays in this enzyme were gen0.001 A OD 340“,J.s. The activity at pH 7.8 was about erally performed at pH 8 and/or 0.1 M KC1 twice that at pH 7.0. The R,,, is the average of at least (3, 14-16). The studies carried out here four determinations. were at a more physiological pH and no additional salt. It should be noted that sufobserved when the reaction was initiated ficient K+ (0.05 M) was present to activate with pyruvate. In fact, at pH 7.8 and added the enzyme and little difference was seen in 0.1 M KCl, no pyruvate induced lag was total activity between 0.05 and 0.15 M K+. observed. In the presence of added 0.1 M Presence of the added salt in the assay KCl, the reaction progress curves generated causes a decrease in the transient effect, as by adding MgATP last were linear for at is illustrated by the data of Table II. Thus, least 1 min and the burst transient was not at pH 8 the transient is not as apparent, apparent at either pH 7 and 8. and could be overlooked by the investigator particularly when using coupled enzyme asDISCUSSION say system. Earlier studies on chicken liver, rat liver, The use of the independent assay methand sheep kidney pyruvate carboxylase ods demonstrate that the transient obhave generally shown that the reaction rate served is not an artifact of the coupled TABLE
II
EFFECT OF SALT AND pH ON LAG TRANSIENT
664
YIP AND RUDOLPH
enzyme assay system. Recently, Easterbrook-Smith et al. (19) showed that enzyme bound CO2 was not stoichiometrically transferred to pyruvate. Therefore, it could be possible that the lag was caused by this nonequivalent transfer because both the coupling enzyme assay and [‘4C]CO~ incorporation assay measured oxalacetate production. However, the lag was still observed with [Y-~~P]ATP assay, ruling out the possibility that the nonequivalent transfer was the cause of the lag. There appear to be two transient effects, one induced by substrates and the other induced by acetyl-CoA. The transient effects can be interpreted as a slow transition between two forms of the enzyme with different catalytic activity. The transition may be due to a change in conformation or in the quaternary structure of the protein or both. The effects of MgATP and pyruvate are antagonistic to each other. MgATP is able to induce the enzyme to shift from a more active conformation to a less active one; pyruvate and bicarbonate cause the enzyme to shift from a less active to a more active conformation. The effect of the nucleotide appears to be predominant over the effect of pyruvate at lower MgATP and pyruvate concentrations. The transient induced by acetyl-CoA was studied in more detail. The observations that the transition was pseudo-first order with respect to time at different enzyme concentration (Fig. 4) and the Arrhenius plot was linear with a low energy of activation (Fig. 7) are all consistent with a conformational change. Even though a conformational change is strongly implicated as the mechanism for the slow transient, under conditions where the slow transient was abolished, e.g., when the enzyme was preincubated with acetylCoA in the cuvette and the reaction was initiated by adding all the substrates together, the non-linear dependence of activity versus enzyme concentration (Fig. 5) strongly suggests that an association process takes place. It is not understood what the interaction between conformational change and change in quaternary structure is, and how it will affect the transient and the catalytic rate of the reaction. Taylor et al. (20) have shown that at low enzyme and
salt concentrations similar to those of this study, the enzyme is a tetramer in the presence of acetyl-CoA but a dimer in its absence. Preincubation of the beef liver enzyme abolished the lag. This result differs from that reported on the chicken liver enzyme, in which case the lag was not abolished (18). The difference between the two results may be due to the species difference between the enzymes. The chicken liver enzyme has been shown to differ from the rat liver and beef liver enzyme in immunological properties and catalytic properties (1,
4). In previous reports on rat liver, chicken liver and sheep kidney enzymes and also in the present report on beef liver, the double reciprocal plot with pyruvate as the varied substrate was biphasic. Recently, Easterbrook-Smith et al. (19) presented an interesting study to explain the biphasic plot. They found the biotin-bound carbon dioxide was not stoichiometrically transferred to pyruvate at low pyruvate concentration and the transfer was stoichiometric at higher pyruvate concentration. The results presented in this report on the effect of pyruvate concentration on the conformation of the enzyme are interesting relative to that finding. As shown in Fig. 6, at low pyruvate concentration (less than 1 mu) and saturating MgATP and HC03-, the enzyme did not exhibit a lag (R,p = 1) while at high pyruvate concentration, the initial rate was slower than the final steady rate (Rslf = 0.7). The rate of the transient also exhibited changes around 1 mu pyruvate. Since both R,,f and k’ varied with pyruvate concentration, it is tempting to speculate that the stoichiometry of carbon dioxide transfer to pyruvate may depend on the conformational state of the enzyme, which in turn depends on the pyruvate concentration. At low pyruvate concentrations, the enzyme remains mainly in the less active conformation, which is not able to transfer carbon dioxide efficiently. At high pyruvate concentrations, the enzyme is shifted to a more active conformation, and nearly stoichiometric transfer of carbon dioxide proceeds. Regulatory implications: The transient
BOVINE
LIVER
PYRUVATE
effect reported here has not been shown to take place in uivo. However, there are examples that an enzyme exhibiting transient properties in vitro also exhibited these properties in vivo (e.g. yeast hexokinase (21)). Thus, the hysteretic properties of the enzyme may be a part of its regulatory mechanisms. In general, two factors are considered to be the primary mechanism for regulation of this enzyme (18, 22). These are acetyl-CoA activation (governing the amount of active enzyme) and the amount of available pyruvate. The introduction of the concept of hysteretic enzyme clearly adds another dimension to the metabolic regulation of the enzyme. According to Frieden (5), a hysteretic enzyme can function as a time-dependent buffer while at the same time slowly responding to changes which will eventually alter the kinetic characteristics of the enzyme to correspond to the altered level of metabolites in the cell. The buffering capacity of such an enzyme system, which depends upon the rate of conversion of one form of the enzyme to another, serves as a mechanism to prevent immediate changes in the concentration of other metabolites in the particular pathway, thus allowing the rate of other intersecting pathways which use a common starting metabolite to be maintained. Both pyruvate and oxalacetate participate in a complexity of metabolic pathways, and it is logical that their levels are tightly regulated in this manner. It is interesting that other enzymes which act on pyruvate are known to exhibit a lag transient. These include phosphoenolpyruvate carboxylase (listed in (23)) and pyruvate kinase (B), suggesting the importance of this mode of control for pyruvate metabolism in vivo. It is also interesting to point out that, to the authors’ knowledge, this is the first demonstration that an enzyme exhibits both a burst and a lag transient depending on the conditions of the assay. ACKNOWLEDGMENTS The authors wish to thank Dr. Scott Power for assistance in the use of the Interdata computer which
CARBOXYLASE
TRANSIENTS
was kindly made available mer.
665
by Professor Graham Pal-
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