Comp, Biochem. Physiol.. 1975. Vol. 52B. pp. 193 to 199. Perffamon Press. Printed in Great Britain
SQUID MUSCLE CITRATE SYNTHASE: CONTROL OF CARBON ENTRY INTO THE KREBS CYCLE P. W. HOCHACHKA,K. B. STOREY 1 and J. BALDWIN Department of Zoology, University of British Columbia, Vancouver, B.C., Canada V6T 1W5 and The Research School of Biological Sciences, Australian National University, Canberra, Australia (Received 11 October 1974) Abstract--1. In squid mantle muscle, citrate synthase occurs in high specific activity as a large 250,000 MW multimeric enzyme. 2. The dnzyme activity is extremely sensitive to NADH inhibition, which is partially reversed by AMP. NADH causes a drastic reduction in the enzyme affinity for oxaloacetate. 3. Squid mantle citrate synthase is also sensitive to inhibition by ATP, c~-ketoglutarate and citrate. ATP effects are competitive with respect to actylCoA while citrate and c~-ketoglutarate inhibit the enzyme by competing with oxaloacetate. 4. These regulatory properties are largely unaffected by pressure.
INTRODUCTION (ct-KGA) and succinylCoA further along the pathway AS FAR as we can tell from available evidence, mantle (Hathaway & Atkinson, 1965; Wright et al., 1967; muscle of the fast swimming squid, Symplectoteuthis Smith & Williamson, 1971). NADH is also an estaboualaniensis, possesses a highly aerobic metabolic lished citrate synthase inhibitor (Srere & Matsuoka, organization with carbohydrate as the main carbon 1972), but the K~ (1.7mM) is about 10-fold higher and energy source (Hochachka et al., 1975). Based than currently accepted estimates of intracellular conon an ~-glycero-P cycle that renders energy metabo- centrations (Edington, 1970); hence in mammalian tislism obligatorily aerobic (Storey & Hochachka, sues, NADH inhibition of citrate synthase is consi1975a) and sustained by a structurally well organized dered of minor physiological significance. and large mass of mitochondria (Moon & Hulbert, In squid mantle muscle, citrate synthase occurs in 1975), this muscle is able to achieve an extremely im- high specific activity as a large, 250,000 MW multipressive step-up in metabolic rate during burst swim- meric enzyme. Its activity appears to be rather more ming occurring in prey capture and predator escape. stringently controlled than in vertebrate tissues We know from. studies of key enzymes in glucose because it is extremely responsive to the redox state metabolism (Storey & Hochachka, 1975b,c,d) that as signalled by NADH/NAD ratios, as well as being these abilities correlate with a novel set of control under the influence (a) of the energy charge (ATP mechanisms in an aerobic glycolysis. But in energetic inhibition; AMP reversal of ATP and NADH effects), terms it is worth emphasizing, aerobic glycolysis and (b) of the degree of Krebs cycle activation, ~merely sets the stage--by producing pyruvate as sub- KGA and citrate both being competitive inhibitors strate--for the final phases of oxidative metabolism. of the enzyme. Inhibition of the enzyme by NADH, As glucose derived pyruvate is the primary and although occurring at concentrations well within the perhaps only significant source of acetylCoA for the physiological range, is complex. At 10pM NADH, Krebs cycle, control of acetylCoA entry into the Krebs for example, enzyme-OXA affinity is reduced by cycle is of crucial significance in sustaining the wide about 20-fold while the enzyme affinity for acetylCoA low-~high activity swings that characterize this is largely unaffected. ATP effects, by contrast, appear tissue. Central to this question of carbon entry into to be strictly competitive with respect to acetylCoA, the Krebs cycle is the regulatory nature of citrate while ~-KGA and citrate inhibit by competing with synthase (E.C.4.1.3.7), catalyzing the first step of the OXA. These regulatory properties are largely unafKrebs cycle: fected by high pressure, although the reaction rate is somewhat pressure inhibited, proceeding with a acetylCoA + OXA--~ CoA + citrate volume change of activation of 8 cm3/mole at 25°C. The latter features are potentially adaptive in a vertiIn mammalian systems, citrate synthase is a well cally migrating organism whose metabolic rate might developed, two-subunit enzyme of about 100,000 MW (Singh et al., 1970; Wu & Yang, 1970), whose activity be depressed during daily deep diving.
is regulated (a) by the energy charge of the cell (ATP inhibition; AMP deinhibition), and (b) by negative feedback from intermediates such as ~-ketoglutarate * Present address: Department of Zoology, Duke University, Durham, NC, U.S.A. 193
MATERIALS AND METHODS
Experimental animals The squid, Symplectoteuthis oualaniensis, was obtained by squid jigging at night in deep waters (surface temperature of 26°C) off the Kona Coast of Hawaii. Upon capture,
194
P.W. HOCHACHKA,K. B. STOREYAND J. BALDWIN
the mantle was quickly excised, freed of any attached tissues, particularly the pigmented epidermis, then chopped into small pieces in preparation for homogenization. Tissue and enzyme preparation Chopped up mantle muscle was homogenized in distilled water (usually 1:10 on a weight/volume basis) for several minutes using a Virtis homogenizer. The homogenate was then spun at 12,000 x g for about 10min. Most of the tissue citrate synthase is solubilized by this procedure; repeated extractions of the precipitate led only to a modest increase in the yield of active enzyme. Three characteristics of this enzyme made purification relatively easy. Firstly, it is fairly stable to short heat exposures; secondly, it is not salted out until quite high ammonium sulphate concentrations are reached; thirdly, it is not strongly absorbed to phosphocellulose at neutral pH. The first step in the purification procedure involved a 47°C heat step for a 3 min period. The solution was then re-chilled in an ice bath at 4°C, spun at about 12,000 x g for 5 min, and the pellet was discarded. The remaining solution was brought to 55% saturation with ammonium sulphate, stirred at 4°C for 30-60min, then centrifuged again at 12,000 x g. The pellet containing less than 10% of the citrate synthase activity was discarded, while the supernatant was brought to 65% saturation with ammonium sulphate. Most of the citrate synthase activity precipitated out in this step. The enzyme was redissolved in small quantities of 50 mM TrisHC1 buffer usually at pH 7.2 and contaminating proteins were further removed by differential absorption onto phosphocellulose as described elsewhere (Hochachka et al., 1975). Our best preparations yielded citrate synthase with a specific activity of about 20 #M product/min per mg protein, compared to 50 for pigeon breast, 40 for moth muscle, 15 for rat liver, and 180 for pig heart (see Singh et al., 1970; Stere, 1969). The purified enzyme was fairly stable on storage in ammonium sulphate, the activity decreasing by about 1/3 after 2 weeks storage. The enzyme was notably free of malate dehydrogenase and lactate dehydrogenase activities as well as of pyruvate kinase, NAD-linked isocitrate dehydrogenase, G6P dehydrogenase, phosphohexose isomerase, and acetylCoA deacetylase. It retained NADP-linked isocitrate dehydrogenase and some fructose-l,6-diphosphatase activity, presumably because all 3 enzymes show a similarly low binding to phosphocellulose under our experimental conditions. Reagents and coupling enzymes All reagents and coupling enzymes were obtained from Sigma Chemical Co., St. Louis, Mo., and unless otherwise specified were reagent grade. Enzyme assay Citrate synthase activity was assayed utilizing the DTNB assay system (Srere, 1969). Optimal conditions required 0-01 mM acetylCoA, 0.134mM OXA, and 0"2 mM DTNB. Effects of pressure on enzyme activity and on regulatory properties were assessed utilizing a temperature controlled high pressure cell built into an SP 1800 Unicam recording spectrophotometer (Mustafa et al., 1971). RESULTS AND DISCUSSION
Molecular size As we indicated above, vertebrate citrate synthases are typically two-subunit enzymes of about 100,000 molecular weight (Wu & Yang, 1970; Singh et al., 1970). In contrast, the squid enzyme behaves on G200 Sephadex columns as if it were a multimeric enzyme of about 250,000 molecular weight (Fig. 1). The same behaviour on G-200 is observed repeatedly, whether a purified or a crude enzyme preparation is
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Fig, 1. A plot of Vo/Ve (void volume/elution volume) vs log mol. wt for a series of proteins of known mol. wt and for squid mantle citrate synthase. G-200 Sephadex was equilibrated with either 30 mM phosphate buffer, pH 8.0 or 50mM Tris-HCl buffer, pH 8.5, and poured onto a column 73 × 2.5cm. Bed volume was 344ml; void volume was 109 ml. Gel filtration was at 4°C and Iatm of pressure.
used in the filtration experiment. Squid citrate synthase is thus seen to resemble certain bacterial citrate synthases (Weitzman & Dunmore, 1969) and like them is highly sensitive to N A D H inhibition (discussed further below). In contrast, the smaller size of the vertebrate enzyme appears to correlate with a greatly reduced N A D H sensitivity (Srere & Matsuoka, 1971). Effect o f pH All animal citrate synthases that have been described to date display alkaline pH optima, usually about pH 8.5-9.0 (Srere, 1969; Hochachka & Lewis, 1970). A similar pH dependence is seen for squid citrate synthase (Fig. 2) except that the pH profile is less steep and there is a broad range through which the enzyme activity is largely pH independent. Effect of temperature and pressure As the squid under study is a vertical migrant, it was important to gain some assessment of the effect of temperature and pressure on the maximum velocity catalyzed by citrate synthase. In regard to temperature, the enzyme appears similar to its homologue in other fishes (Hochachka & Lewis, 1970) displaying
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Fig. 2. The pH requirements for squid citrate synthase at 25°C, 14.7 psi, and saturating concentrations of both substrates.
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concentrations, 50 mM Tris-HCl buffer, pH 7.8 at low and high temperatures. linear Arrhenius plots between 5°. and 30°C and a Q10 of about 1.5 compared to about 1-7 for the trout liver enzyme. Th~ effect of pressure, by comparison, is modest (Fig. 3). At low (6°C) temperature, the enzyme reaction rate appears to be slightly activated at pressures up to about 6000psi, while at higher pressures it is inhibited. At 25°C, the activation phenomenon is not observed and the reaction proceeds with a small volume change of activation (8 cm3/mole between 1 and 500 atm). Considering the charged nature of substrates and products, the small absolute pressure effect on the reaction is rather remarkable (Weale, 1967); although of obvious biological significance, the mechanistic basis for such a pressure-resistant enzyme is not at all understood. Although not directly comparable to typical vertebrate citrate synthases, squid citrate synthase responds to pressure in a manner comparable to the gill enzyme of the abyssal fish, Antimora (Hoehachka, 1975, this series). Saturation kinetics Squid mantle muscle citrate synthase saturation curves for both OXA and acetylCoA are rectangular hyperbolas. In agreement with previous studies (see Johansson et al., 1973, for. example), the Michaelis constant ( K J for each substrate is independent of the concentration of the cosubstrate. Lineweaver-Burk plots for OXA and for acetylCoA are invariably linear over the relatively narrow concentration ranges used, an observation that also is in accord with previous studies (Matsuoka & Srere, 1973; Johannson et al., 1973). Km values of 1-2 pM for OXA and 2 - 3 / ~ I for acetylCoA are estimated at 25°C and 14.7 psi, indicating very high enzyme affinities for both substrates. Compared to purified pig heart citrate syn-
195
thase, the squid enzyme displays about a 4-fold higher affinity for OXA and about a 3-fold higher affinity for acetylCoA. The K m values obtained for the enzyme in the presence of only its substrates would appear to be lower than probable in vivo concentrations of OXA and acetylCoA, implying either that the enzyme is always fully saturated or that affinity for its substrates is metabolite regulated under in vivo conditions. Examination of the enzyme's regulatory properties indicates that the latter alternative is by far the more likely. In this regard, various workers have demonstrated that the Krebs cycle can be divided operationally into at least two reaction spans, each being independently controlled at least to some degree. The first span consists of the sequence of reactions leading from citrate ~-KGA and in this span citrate synthase, being the first step in the reaction pathway, functions as an important control point (see Randle et al., 1971). In accord with well known principles of biological feedback (see Atkinson, 1966, for example), citrate synthase activity in mammalian and bacterial systems is known to be under negative feedback control by two kinds of metabolites: firstly, Krebs cycle intermediates, such as ct-KGA (Wright et al., 1967) or succinylCoA (Smith & Williamson, 1971), serve as metabolic signals of the degree of augmentation of cycle intermediates during Krebs cycle activation and they therefore integrate citrate synthase activity directly with the rest of the Krebs cycle. And secondly, the concentrations of the adenylates serve as metabolic signals of the overall energy status of the cell and hence supply a sensitive measure of the degree to which Krebs cycle function is required (Atkinson, 1968; Garland, 1968). In the above context, therefore, we were not surprised to find that the activity of squid mantle muscle citrate synthase also is strongly influenced by Krebs cycle intermediates and by the adenylates, although the mechanism of action of these two classes of inhibitors is different. Regulation by Krebs cycle intermediates All of the Krebs cycle intermediates were tested as potential citrate synthase .modulators, except succinylCoA, which interfered with our assay because of contaminants reacting with DTNB. Within this cycle, only ~-KGA and citrate were found to have a strongly inhibitory effect on squid muscle citrate synthase. The mechanism of action of citrate and ~-KGA is complex. With respect to OXA, both compounds are strictly competitive inhibitors, causing large decreases in enzyme-OXA affinity with no change in the calculated Vmax(Fig. 4). In contrast, with respect tO acetylCoA, citrate and ~-KGA effects are largely on the Vmax with enzyme-acetylCoA affinity being unaffected by ~-KGA but slightly decreased with citrate (Fig. 5), a result in contrast with similar experiments on the mammalian enzyme (Matsuoka & Srere, 1973). Whereas one might expect large volume changes to occur during citrate synthase binding of strongly charged compounds such as ct-KGA or citrate, this does not appear to be the case for high pressure does not affect the inhibition curve (Fig. 6). Since ~t-KGA acts in a manner competitive with OXA, this result indicates unequivocally that enzyme-OXA affinity also
196
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Fig. 4. Double reciprocal plots (l/velocity vs I/OXA concentration) for squid mantle citrate synthase in the presence and absence of 20 mM citrate. Assay conditions: 0.01 mM acetylCoA, varying OXA concentrations, pH 7.5, 50mM Tris-HC1 buffer, at 25°C and 14.7psi. Inset on the left shows the saturation curves in the presence and absence of 20 mM citrate. Effects of 7-KGA at concentrations of 4-10 mM lead to comparable inhibition.
is pressure independent. (Operationally such a conclusion is important since the lag time, between initiating reaction and establishing working high pressure, with our current methods of high pressure spectrophotometry do not allow accurate measures at high pressure of citrate synthase reaction rates at low OXA concentrations, and hence do not allow a direct assessment of the effect of pressure on the K,, for this substrate.) Especial significance is attached to the effects of ~-KGA and citrate for two reasons. Firstly, 7-KGA represents the "end product" of the first span of the Krebs cycle and its concentration is a potentially important measure of the degree to which intermediates in the second span of the Krebs cycle (c~-KGA---~ OXA) are augmented during activation of catabolism. It is important to emphasize that this "information" could not be supplied to the citrate synthase control site by any intermediate formed prior to a-KGA in the first span of the cycle because 7-KGA can arise not only ,from citrate but also from glutamate (either by
transamination reactions or by glutamate dehydrogenase). In this context, nature's choice of c~-KGA as one mechanism of controlling carbon entry into the Krebs cycle can be fully appreciated. A slightly different situation holds with regard to citrate inhibition. In squid mantle muscle, citrate appears to play an important role in integrating aerobic glycolysis with the activity of the Krebs cycle, by its inhibitory actions on phosphofructokinase (Storey & Hochachka, 1975c) and on pyruvate kinase (Storey & Hochachka, 1975d). A controlling effect on its own further synthesis, then, would appear an imperative condition for closing the control "loop" between aerobic glycolysis and the Krebs cycle.
Regulation by the adenylates The situation with regard to adenylate control of squid muscle citrate synthase is entirely comparable to that already observed for the homologous enzyme from other sources (see Atkinson, 1968; Garland, 1968). Concentrations of ATP within the expected
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Fig. 5. AcetylCoA saturation curves (left panel) in the presence and absence of 20 mM citrate for squid muscle citrate synthase at pH 7-5, 50mM Tris-HCl, at 25°C, 14.7 psi, with saturating concentrations of OXA and varying acetylCoA levels. Lineweaver-Burk plots of the data are shown in the right panel. At 4 10mM levels of :~-KGA, a similar percentage inhibition is obtained that is strictly non-competitive with respect to acetylCoA; i.e. :~-KGA, unlike citrate, does not alter the Km for acetylCoA.
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Fig. 6. Effect of pressure on ~-KGA inhibition curves for squid muscle citrate synthase at 5°C assayed at 0.04 mM acetylCoA, 0.04mM OXA, pH 7.8, 50mM Tris-HC1 buffer. Reaction velocity is in E412/4 min. physiological range are strongly inhibitory, while AMP and ADP at physiological levels have little or no effect on the enzyme activity. ATP inhibition is competitive with respect to acetylCoA (Fig. 7), 2 mM levels causing about a 5-fold increase in the K m for acetylCoA. As ATP is the biologically most important "end product" of energy metabolism, ATP inhibition at this enzyme locus is readily appreciated. At first impression, the mechanism of ATP inhibition appears relatively simple; it is in fact complex. As we argued above, enzyme binding of an inhibitor as highly charged as ATP should proceed with large volume changes and therefore the inhibitory effect should be strongly influenced by pressure. For squid citrate synthase that is not the case. ATP inhibition curves are essentially identical at both low and high pressures (Fig. 8), whether the experiment is done at low (5°C) or at high (25°C) temperatures. In biochemical terms, what this result undoubtedly means is that enzyme affinity for acetyICoA as well as for
its competitive inhibitor, ATP, is totally pressure independent. In metabolic terms, it means that the competitive ability of citrate synthase for either of its substrates, OXA or acetylCoA, is similar at all pressures that are encountered in nature. Furthermore, the adenylate and metabolite signals that alter enzyme affinities for its two substrates, themselves appear to
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~¢~eau0x'~ Fig. 7. Double reciprocal plots (1/velocity vs 1/acetylCoA concentration) for squid muscle citrate synthase in the absence and presence of 2 mM ATP at 25°C, 14-7 psi, 0.2 mM OXA, pH 7-5. ATP increases the Km for acetylCoA greatly but has no effect on the calculated Vmax.
Fig. 8. Effect of pressure on the ATP inhibition curve for squid citrate synthase at 5°C, 0.04mM acetylCoA, 0"04 mM OXA, pH 7.8, 50 mM Tris-HCl buffer. Inhibition at 14.7 psi is shown in circles; at 10,000psi, in squares. Reaction velocity (v) in E412/4min. be equally effective at all pressures examined. Such pressure independence of enzyme regulatory properties appears to be an imperative feature of metabolic control in abyssal and vertically migrating organisms (Hochachka et al., 1971; Moon et al., 1971) and it is also observed in our. studies of an extremely potent NADH inhibition of the squid citrate synthase (Fig. 9).
Redox regulation of squid citrate synthase As we have emphasized above, perhaps the most novel control feature of aerobic glycolysis in the squid mantle muscle is the pivotal role played by NADH. It is involved in inhibitory control at least of 3 important glycolytic enzymes---phosphofructokinase, pyruvate kinase, and glyceraldehyde-3-P dehydrogenase (Storey & Hochachka, 1975b,c,d). During whole-animal states of reduced metabolic rate, at the end of deep diving, for example, a high energy charge is thought to hold cytoplasmic ~-GPDH in an inhibited state while NADH along with other effectors is thought to dampen glycolytic rates. Transition to higher work rates leads to a drop in ATP concentrations, a sparking of cytoplasmic ~-GPDH, and an automatic release of glycolysis from NADH inhibition. In this view NADH is at the hub of cytoplasmic control circuitry because it closely integrates the cytoplasmic arm of the ~-GP cycle with glycolytic activation. Most instructive, in this context, is the observation that through NADH inhibition of citrate synthase the mitochondrial arm of the ~-GP cycle (and thus the activity of the electron transfer system per se) is closely coordinated with the rate of carbon entry into the Krebs cycle. Of all regulatory effectors found for squid muscle citrate synthase, NADH is undoubtedly the most potent, with an apparent K~ of about 20 #M (Fig. 9). The mechanism of inhibition is surprisingly complex. By structural analogy, one would anticipate NADH inhibition to mimic ATP inhibition in being competitive with respect to acetylCoA (Srere & Matsuoka, 1971), but in fact we observed that the K m for acetylCoA is essentially unchanged in the presence of 0.01 mM NADH. In sharp contrast, NADH at similarly low concentrations dramatically increases the apparent Km for OXA by 20- to 25-fold (Fig. 10).
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SQUID MUSCLE CITRATE SYNTHASE: CONTROL OF CARBON ENTRY INTO THE KREBS CYCLE P. W. HOCHACHKA,K. B. STOREY 1 and J. BALDWIN Department of Zoology, University of British Columbia, Vancouver, B.C., Canada V6T 1W5 and The Research School of Biological Sciences, Australian National University, Canberra, Australia (Received 11 October 1974) Abstract--1. In squid mantle muscle, citrate synthase occurs in high specific activity as a large 250,000 MW multimeric enzyme. 2. The dnzyme activity is extremely sensitive to NADH inhibition, which is partially reversed by AMP. NADH causes a drastic reduction in the enzyme affinity for oxaloacetate. 3. Squid mantle citrate synthase is also sensitive to inhibition by ATP, c~-ketoglutarate and citrate. ATP effects are competitive with respect to actylCoA while citrate and c~-ketoglutarate inhibit the enzyme by competing with oxaloacetate. 4. These regulatory properties are largely unaffected by pressure.
INTRODUCTION (ct-KGA) and succinylCoA further along the pathway AS FAR as we can tell from available evidence, mantle (Hathaway & Atkinson, 1965; Wright et al., 1967; muscle of the fast swimming squid, Symplectoteuthis Smith & Williamson, 1971). NADH is also an estaboualaniensis, possesses a highly aerobic metabolic lished citrate synthase inhibitor (Srere & Matsuoka, organization with carbohydrate as the main carbon 1972), but the K~ (1.7mM) is about 10-fold higher and energy source (Hochachka et al., 1975). Based than currently accepted estimates of intracellular conon an ~-glycero-P cycle that renders energy metabo- centrations (Edington, 1970); hence in mammalian tislism obligatorily aerobic (Storey & Hochachka, sues, NADH inhibition of citrate synthase is consi1975a) and sustained by a structurally well organized dered of minor physiological significance. and large mass of mitochondria (Moon & Hulbert, In squid mantle muscle, citrate synthase occurs in 1975), this muscle is able to achieve an extremely im- high specific activity as a large, 250,000 MW multipressive step-up in metabolic rate during burst swim- meric enzyme. Its activity appears to be rather more ming occurring in prey capture and predator escape. stringently controlled than in vertebrate tissues We know from. studies of key enzymes in glucose because it is extremely responsive to the redox state metabolism (Storey & Hochachka, 1975b,c,d) that as signalled by NADH/NAD ratios, as well as being these abilities correlate with a novel set of control under the influence (a) of the energy charge (ATP mechanisms in an aerobic glycolysis. But in energetic inhibition; AMP reversal of ATP and NADH effects), terms it is worth emphasizing, aerobic glycolysis and (b) of the degree of Krebs cycle activation, ~merely sets the stage--by producing pyruvate as sub- KGA and citrate both being competitive inhibitors strate--for the final phases of oxidative metabolism. of the enzyme. Inhibition of the enzyme by NADH, As glucose derived pyruvate is the primary and although occurring at concentrations well within the perhaps only significant source of acetylCoA for the physiological range, is complex. At 10pM NADH, Krebs cycle, control of acetylCoA entry into the Krebs for example, enzyme-OXA affinity is reduced by cycle is of crucial significance in sustaining the wide about 20-fold while the enzyme affinity for acetylCoA low-~high activity swings that characterize this is largely unaffected. ATP effects, by contrast, appear tissue. Central to this question of carbon entry into to be strictly competitive with respect to acetylCoA, the Krebs cycle is the regulatory nature of citrate while ~-KGA and citrate inhibit by competing with synthase (E.C.4.1.3.7), catalyzing the first step of the OXA. These regulatory properties are largely unafKrebs cycle: fected by high pressure, although the reaction rate is somewhat pressure inhibited, proceeding with a acetylCoA + OXA--~ CoA + citrate volume change of activation of 8 cm3/mole at 25°C. The latter features are potentially adaptive in a vertiIn mammalian systems, citrate synthase is a well cally migrating organism whose metabolic rate might developed, two-subunit enzyme of about 100,000 MW (Singh et al., 1970; Wu & Yang, 1970), whose activity be depressed during daily deep diving.
is regulated (a) by the energy charge of the cell (ATP inhibition; AMP deinhibition), and (b) by negative feedback from intermediates such as ~-ketoglutarate * Present address: Department of Zoology, Duke University, Durham, NC, U.S.A. 193
MATERIALS AND METHODS
Experimental animals The squid, Symplectoteuthis oualaniensis, was obtained by squid jigging at night in deep waters (surface temperature of 26°C) off the Kona Coast of Hawaii. Upon capture,
194
P.W. HOCHACHKA,K. B. STOREYAND J. BALDWIN
the mantle was quickly excised, freed of any attached tissues, particularly the pigmented epidermis, then chopped into small pieces in preparation for homogenization. Tissue and enzyme preparation Chopped up mantle muscle was homogenized in distilled water (usually 1:10 on a weight/volume basis) for several minutes using a Virtis homogenizer. The homogenate was then spun at 12,000 x g for about 10min. Most of the tissue citrate synthase is solubilized by this procedure; repeated extractions of the precipitate led only to a modest increase in the yield of active enzyme. Three characteristics of this enzyme made purification relatively easy. Firstly, it is fairly stable to short heat exposures; secondly, it is not salted out until quite high ammonium sulphate concentrations are reached; thirdly, it is not strongly absorbed to phosphocellulose at neutral pH. The first step in the purification procedure involved a 47°C heat step for a 3 min period. The solution was then re-chilled in an ice bath at 4°C, spun at about 12,000 x g for 5 min, and the pellet was discarded. The remaining solution was brought to 55% saturation with ammonium sulphate, stirred at 4°C for 30-60min, then centrifuged again at 12,000 x g. The pellet containing less than 10% of the citrate synthase activity was discarded, while the supernatant was brought to 65% saturation with ammonium sulphate. Most of the citrate synthase activity precipitated out in this step. The enzyme was redissolved in small quantities of 50 mM TrisHC1 buffer usually at pH 7.2 and contaminating proteins were further removed by differential absorption onto phosphocellulose as described elsewhere (Hochachka et al., 1975). Our best preparations yielded citrate synthase with a specific activity of about 20 #M product/min per mg protein, compared to 50 for pigeon breast, 40 for moth muscle, 15 for rat liver, and 180 for pig heart (see Singh et al., 1970; Stere, 1969). The purified enzyme was fairly stable on storage in ammonium sulphate, the activity decreasing by about 1/3 after 2 weeks storage. The enzyme was notably free of malate dehydrogenase and lactate dehydrogenase activities as well as of pyruvate kinase, NAD-linked isocitrate dehydrogenase, G6P dehydrogenase, phosphohexose isomerase, and acetylCoA deacetylase. It retained NADP-linked isocitrate dehydrogenase and some fructose-l,6-diphosphatase activity, presumably because all 3 enzymes show a similarly low binding to phosphocellulose under our experimental conditions. Reagents and coupling enzymes All reagents and coupling enzymes were obtained from Sigma Chemical Co., St. Louis, Mo., and unless otherwise specified were reagent grade. Enzyme assay Citrate synthase activity was assayed utilizing the DTNB assay system (Srere, 1969). Optimal conditions required 0-01 mM acetylCoA, 0.134mM OXA, and 0"2 mM DTNB. Effects of pressure on enzyme activity and on regulatory properties were assessed utilizing a temperature controlled high pressure cell built into an SP 1800 Unicam recording spectrophotometer (Mustafa et al., 1971). RESULTS AND DISCUSSION
Molecular size As we indicated above, vertebrate citrate synthases are typically two-subunit enzymes of about 100,000 molecular weight (Wu & Yang, 1970; Singh et al., 1970). In contrast, the squid enzyme behaves on G200 Sephadex columns as if it were a multimeric enzyme of about 250,000 molecular weight (Fig. 1). The same behaviour on G-200 is observed repeatedly, whether a purified or a crude enzyme preparation is
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Fig, 1. A plot of Vo/Ve (void volume/elution volume) vs log mol. wt for a series of proteins of known mol. wt and for squid mantle citrate synthase. G-200 Sephadex was equilibrated with either 30 mM phosphate buffer, pH 8.0 or 50mM Tris-HCl buffer, pH 8.5, and poured onto a column 73 × 2.5cm. Bed volume was 344ml; void volume was 109 ml. Gel filtration was at 4°C and Iatm of pressure.
used in the filtration experiment. Squid citrate synthase is thus seen to resemble certain bacterial citrate synthases (Weitzman & Dunmore, 1969) and like them is highly sensitive to N A D H inhibition (discussed further below). In contrast, the smaller size of the vertebrate enzyme appears to correlate with a greatly reduced N A D H sensitivity (Srere & Matsuoka, 1971). Effect o f pH All animal citrate synthases that have been described to date display alkaline pH optima, usually about pH 8.5-9.0 (Srere, 1969; Hochachka & Lewis, 1970). A similar pH dependence is seen for squid citrate synthase (Fig. 2) except that the pH profile is less steep and there is a broad range through which the enzyme activity is largely pH independent. Effect of temperature and pressure As the squid under study is a vertical migrant, it was important to gain some assessment of the effect of temperature and pressure on the maximum velocity catalyzed by citrate synthase. In regard to temperature, the enzyme appears similar to its homologue in other fishes (Hochachka & Lewis, 1970) displaying
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Fig. 2. The pH requirements for squid citrate synthase at 25°C, 14.7 psi, and saturating concentrations of both substrates.
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Fig. 3. Effect of pressure on the reaction velocity catalyzed by squid muscle citrate synthas¢ under saturating substrate
concentrations, 50 mM Tris-HCl buffer, pH 7.8 at low and high temperatures. linear Arrhenius plots between 5°. and 30°C and a Q10 of about 1.5 compared to about 1-7 for the trout liver enzyme. Th~ effect of pressure, by comparison, is modest (Fig. 3). At low (6°C) temperature, the enzyme reaction rate appears to be slightly activated at pressures up to about 6000psi, while at higher pressures it is inhibited. At 25°C, the activation phenomenon is not observed and the reaction proceeds with a small volume change of activation (8 cm3/mole between 1 and 500 atm). Considering the charged nature of substrates and products, the small absolute pressure effect on the reaction is rather remarkable (Weale, 1967); although of obvious biological significance, the mechanistic basis for such a pressure-resistant enzyme is not at all understood. Although not directly comparable to typical vertebrate citrate synthases, squid citrate synthase responds to pressure in a manner comparable to the gill enzyme of the abyssal fish, Antimora (Hoehachka, 1975, this series). Saturation kinetics Squid mantle muscle citrate synthase saturation curves for both OXA and acetylCoA are rectangular hyperbolas. In agreement with previous studies (see Johansson et al., 1973, for. example), the Michaelis constant ( K J for each substrate is independent of the concentration of the cosubstrate. Lineweaver-Burk plots for OXA and for acetylCoA are invariably linear over the relatively narrow concentration ranges used, an observation that also is in accord with previous studies (Matsuoka & Srere, 1973; Johannson et al., 1973). Km values of 1-2 pM for OXA and 2 - 3 / ~ I for acetylCoA are estimated at 25°C and 14.7 psi, indicating very high enzyme affinities for both substrates. Compared to purified pig heart citrate syn-
195
thase, the squid enzyme displays about a 4-fold higher affinity for OXA and about a 3-fold higher affinity for acetylCoA. The K m values obtained for the enzyme in the presence of only its substrates would appear to be lower than probable in vivo concentrations of OXA and acetylCoA, implying either that the enzyme is always fully saturated or that affinity for its substrates is metabolite regulated under in vivo conditions. Examination of the enzyme's regulatory properties indicates that the latter alternative is by far the more likely. In this regard, various workers have demonstrated that the Krebs cycle can be divided operationally into at least two reaction spans, each being independently controlled at least to some degree. The first span consists of the sequence of reactions leading from citrate ~-KGA and in this span citrate synthase, being the first step in the reaction pathway, functions as an important control point (see Randle et al., 1971). In accord with well known principles of biological feedback (see Atkinson, 1966, for example), citrate synthase activity in mammalian and bacterial systems is known to be under negative feedback control by two kinds of metabolites: firstly, Krebs cycle intermediates, such as ct-KGA (Wright et al., 1967) or succinylCoA (Smith & Williamson, 1971), serve as metabolic signals of the degree of augmentation of cycle intermediates during Krebs cycle activation and they therefore integrate citrate synthase activity directly with the rest of the Krebs cycle. And secondly, the concentrations of the adenylates serve as metabolic signals of the overall energy status of the cell and hence supply a sensitive measure of the degree to which Krebs cycle function is required (Atkinson, 1968; Garland, 1968). In the above context, therefore, we were not surprised to find that the activity of squid mantle muscle citrate synthase also is strongly influenced by Krebs cycle intermediates and by the adenylates, although the mechanism of action of these two classes of inhibitors is different. Regulation by Krebs cycle intermediates All of the Krebs cycle intermediates were tested as potential citrate synthase .modulators, except succinylCoA, which interfered with our assay because of contaminants reacting with DTNB. Within this cycle, only ~-KGA and citrate were found to have a strongly inhibitory effect on squid muscle citrate synthase. The mechanism of action of citrate and ~-KGA is complex. With respect to OXA, both compounds are strictly competitive inhibitors, causing large decreases in enzyme-OXA affinity with no change in the calculated Vmax(Fig. 4). In contrast, with respect tO acetylCoA, citrate and ~-KGA effects are largely on the Vmax with enzyme-acetylCoA affinity being unaffected by ~-KGA but slightly decreased with citrate (Fig. 5), a result in contrast with similar experiments on the mammalian enzyme (Matsuoka & Srere, 1973). Whereas one might expect large volume changes to occur during citrate synthase binding of strongly charged compounds such as ct-KGA or citrate, this does not appear to be the case for high pressure does not affect the inhibition curve (Fig. 6). Since ~t-KGA acts in a manner competitive with OXA, this result indicates unequivocally that enzyme-OXA affinity also
196
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Fig. 4. Double reciprocal plots (l/velocity vs I/OXA concentration) for squid mantle citrate synthase in the presence and absence of 20 mM citrate. Assay conditions: 0.01 mM acetylCoA, varying OXA concentrations, pH 7.5, 50mM Tris-HC1 buffer, at 25°C and 14.7psi. Inset on the left shows the saturation curves in the presence and absence of 20 mM citrate. Effects of 7-KGA at concentrations of 4-10 mM lead to comparable inhibition.
is pressure independent. (Operationally such a conclusion is important since the lag time, between initiating reaction and establishing working high pressure, with our current methods of high pressure spectrophotometry do not allow accurate measures at high pressure of citrate synthase reaction rates at low OXA concentrations, and hence do not allow a direct assessment of the effect of pressure on the K,, for this substrate.) Especial significance is attached to the effects of ~-KGA and citrate for two reasons. Firstly, 7-KGA represents the "end product" of the first span of the Krebs cycle and its concentration is a potentially important measure of the degree to which intermediates in the second span of the Krebs cycle (c~-KGA---~ OXA) are augmented during activation of catabolism. It is important to emphasize that this "information" could not be supplied to the citrate synthase control site by any intermediate formed prior to a-KGA in the first span of the cycle because 7-KGA can arise not only ,from citrate but also from glutamate (either by
transamination reactions or by glutamate dehydrogenase). In this context, nature's choice of c~-KGA as one mechanism of controlling carbon entry into the Krebs cycle can be fully appreciated. A slightly different situation holds with regard to citrate inhibition. In squid mantle muscle, citrate appears to play an important role in integrating aerobic glycolysis with the activity of the Krebs cycle, by its inhibitory actions on phosphofructokinase (Storey & Hochachka, 1975c) and on pyruvate kinase (Storey & Hochachka, 1975d). A controlling effect on its own further synthesis, then, would appear an imperative condition for closing the control "loop" between aerobic glycolysis and the Krebs cycle.
Regulation by the adenylates The situation with regard to adenylate control of squid muscle citrate synthase is entirely comparable to that already observed for the homologous enzyme from other sources (see Atkinson, 1968; Garland, 1968). Concentrations of ATP within the expected
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Fig. 5. AcetylCoA saturation curves (left panel) in the presence and absence of 20 mM citrate for squid muscle citrate synthase at pH 7-5, 50mM Tris-HCl, at 25°C, 14.7 psi, with saturating concentrations of OXA and varying acetylCoA levels. Lineweaver-Burk plots of the data are shown in the right panel. At 4 10mM levels of :~-KGA, a similar percentage inhibition is obtained that is strictly non-competitive with respect to acetylCoA; i.e. :~-KGA, unlike citrate, does not alter the Km for acetylCoA.
Squid citrate synthase
197
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Fig. 6. Effect of pressure on ~-KGA inhibition curves for squid muscle citrate synthase at 5°C assayed at 0.04 mM acetylCoA, 0.04mM OXA, pH 7.8, 50mM Tris-HC1 buffer. Reaction velocity is in E412/4 min. physiological range are strongly inhibitory, while AMP and ADP at physiological levels have little or no effect on the enzyme activity. ATP inhibition is competitive with respect to acetylCoA (Fig. 7), 2 mM levels causing about a 5-fold increase in the K m for acetylCoA. As ATP is the biologically most important "end product" of energy metabolism, ATP inhibition at this enzyme locus is readily appreciated. At first impression, the mechanism of ATP inhibition appears relatively simple; it is in fact complex. As we argued above, enzyme binding of an inhibitor as highly charged as ATP should proceed with large volume changes and therefore the inhibitory effect should be strongly influenced by pressure. For squid citrate synthase that is not the case. ATP inhibition curves are essentially identical at both low and high pressures (Fig. 8), whether the experiment is done at low (5°C) or at high (25°C) temperatures. In biochemical terms, what this result undoubtedly means is that enzyme affinity for acetyICoA as well as for
its competitive inhibitor, ATP, is totally pressure independent. In metabolic terms, it means that the competitive ability of citrate synthase for either of its substrates, OXA or acetylCoA, is similar at all pressures that are encountered in nature. Furthermore, the adenylate and metabolite signals that alter enzyme affinities for its two substrates, themselves appear to
+
~¢~eau0x'~ Fig. 7. Double reciprocal plots (1/velocity vs 1/acetylCoA concentration) for squid muscle citrate synthase in the absence and presence of 2 mM ATP at 25°C, 14-7 psi, 0.2 mM OXA, pH 7-5. ATP increases the Km for acetylCoA greatly but has no effect on the calculated Vmax.
Fig. 8. Effect of pressure on the ATP inhibition curve for squid citrate synthase at 5°C, 0.04mM acetylCoA, 0"04 mM OXA, pH 7.8, 50 mM Tris-HCl buffer. Inhibition at 14.7 psi is shown in circles; at 10,000psi, in squares. Reaction velocity (v) in E412/4min. be equally effective at all pressures examined. Such pressure independence of enzyme regulatory properties appears to be an imperative feature of metabolic control in abyssal and vertically migrating organisms (Hochachka et al., 1971; Moon et al., 1971) and it is also observed in our. studies of an extremely potent NADH inhibition of the squid citrate synthase (Fig. 9).
Redox regulation of squid citrate synthase As we have emphasized above, perhaps the most novel control feature of aerobic glycolysis in the squid mantle muscle is the pivotal role played by NADH. It is involved in inhibitory control at least of 3 important glycolytic enzymes---phosphofructokinase, pyruvate kinase, and glyceraldehyde-3-P dehydrogenase (Storey & Hochachka, 1975b,c,d). During whole-animal states of reduced metabolic rate, at the end of deep diving, for example, a high energy charge is thought to hold cytoplasmic ~-GPDH in an inhibited state while NADH along with other effectors is thought to dampen glycolytic rates. Transition to higher work rates leads to a drop in ATP concentrations, a sparking of cytoplasmic ~-GPDH, and an automatic release of glycolysis from NADH inhibition. In this view NADH is at the hub of cytoplasmic control circuitry because it closely integrates the cytoplasmic arm of the ~-GP cycle with glycolytic activation. Most instructive, in this context, is the observation that through NADH inhibition of citrate synthase the mitochondrial arm of the ~-GP cycle (and thus the activity of the electron transfer system per se) is closely coordinated with the rate of carbon entry into the Krebs cycle. Of all regulatory effectors found for squid muscle citrate synthase, NADH is undoubtedly the most potent, with an apparent K~ of about 20 #M (Fig. 9). The mechanism of inhibition is surprisingly complex. By structural analogy, one would anticipate NADH inhibition to mimic ATP inhibition in being competitive with respect to acetylCoA (Srere & Matsuoka, 1971), but in fact we observed that the K m for acetylCoA is essentially unchanged in the presence of 0.01 mM NADH. In sharp contrast, NADH at similarly low concentrations dramatically increases the apparent Km for OXA by 20- to 25-fold (Fig. 10).
198
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