Controls of citrate synthase activity

Life Sciences Vol . 15, pp . 1695-1710 Printed in the U .S .A .

Pe rgamon Press

MINIREVIEW

CONTROLS OF CITRATE SYNTHASE ACTIVITY Paul A. Srere Pre-Clinical Science Unit, Veterans Administration Hospital, and Department of Biochemistry, The University of Texas Health Science Center, Dallas, Texas

75216

Sihß"IARY The inhibition of citrate synthase bY a variety of nucleotides and polycarboxylate compounds is not unexpected since many of the compounds are substrate analogs of citrate synthase . These effectors are interesting by virtue of the fact that many of them are intermediates and/or end products in the metabolic path of which citrate synthase can be considered the first committed step . As a consequence, it is possible to propose re lation of citrate synthase by ATP (or phosphorylation potential~by acyl CoA (acylation level) and NADH (redox potential) . Aside from these putative controls, it is possible that the major control of citrate synthase activity is by changes in the concentration of its substrates acetyl CoA and oxalacetate" I discuss in this review the many factors that must be considered before one can decide whether or not interactions between metabolites and enzymes observed in an in vitro catalytic situation have metabolic relevance" These factors include 1) the concentrations of substrates at the enzyme site, 2) the concentrations of effectors at the enzyme site, 3) the presence of modifying substances, and 4) the difference in behavior of an enzyme at its concentration in vivo compared to its concentration in vitro. In the case of citrate synthase as is generally true for other enzymes, no accurate lmowledge of these factors are available in vitro so that little can be said concerning the in situ control of citrate synthase, which may be the result of all the factors acting in concert . The studies of effectors on enzymes in vitro can only serve as a guideline for parameters to study whén techniques are available to study control of enzymes in situ.

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INTRODUCTION Citrate

(si)-synthase

(E .C . ~~ " 1 .3 " 7) is considered the

major regulatory site of Krebs cycle activity by many and recent studies have suggested a wide variety of possible effectors of this enzyme's activity (1) . I describe in this article the multiple ways in which citrate synthase activity can be altered including changes in enzyme amount, changes in putative effectors concentrations and changes in its substrate concentration " Most of the information reported in this short review was obtained from in vitro studies " By their inclusion I do not imply that we know what controls citrate synthase activity or even that we know that citrate synthase activity does control Krebs cycle activity . Other enzymes also have been proposed as the site of regulation of Krebs cycle activity and these include pyruvate dehydrogenase,

isocitrate

dehydrogenase and a-ketoglutarate dehydrogenase " Possible regulatory interactions have been reported between Krebs cycle intermediates and the enzymes aconitase, ase and malate dehydrogenase .

succincte dehydrogenase,

fumer-

In a recent book on metabolic

regulation, Newsho]me and Start (2 ),

after proposing regulation

of "the early stages" of Krebs cycle activity at citrate synthase and isocitrate dehydrogenase, go on to state that there is insufficient evidence about the regulation to come to an acceptable conclusion " I have pointed out earlier

(3) that the complexity of

most metabolic systems makes it unlikely that control will occur at a single point in a pathway "

It is possible that one or more

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of the interactions described here is responsible for control of citrate synthase activity which in turn may regulate the rate of Krebs cycle oxidations " However, before we can describe the control of Krebs cycle activity knowledge is needed of the changes of all substrates and effectors at the enzyme sites concomitant with changes in cycle activity . At present there is no method of obtaining a direct measure of the free concentration of cycle intermediates in the mitochondria of intact tissue, let alone knowing about local concentrations of these compounds " These words of caution are included to help prevent overinterpretation of the interactions I have described below. Changes in Amount of En~me Studies on microorganisms show that adaptation to new growth conditions is often accompanied by increases in the rate of synthesis of enzymes" This has been described with citrate synthase in bacteria (~+) and in yeast (5). In animal cells increases in the amount of enzyme can occur either by increased synthesis or by decreased breakdown of the enzyme " One of the best documented cases of increases in an animal citrate synthase is the work of Holloszy et al " (6) who showed that in exercised rats the skeletal muscles showed increases in the Krebs cycle enzymes including citrate synthase " Frenkel et al " (7) have shown that Vitamin H12 deficiency resulted in an increase (2X) of citrate synthase of rat liver and that the number of liver mitochondria and the surface area of their cristae had increased" Citrate synthase activity is not as labile as the enzymes of fatty acid synthesis or gluconeogenesis, since starvation and varied diets

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have little effect on it.s activity (8) . Although it seems certain that citrate synthase (a mitochondrial enzyme) is synthesized in the cytosol no studies on its turnover in animal cells has been reported nor do we know what is responsible for its induction or repression " Krebs cycle enzymes occur in a fairly constant proportion from tissue to tissue so that it seems logical to assume that common mechanisms occur for their induction or repression . Judging from the correlation between Krebs cycle enzyme activity and 02 consumption of a tissue (5), one might conclude that 02 tension within the cell played a key role in induction or repression of citrate synthase and other Krebs cycle enzymes . It is interesting that, unlike some enzymes whose properties vary from tissue to tissue within an animal to accommodate the special metabolism of each tissue, citrate synthase from heart, liver, kidney and brain of the rat seem to be identical (9,10) " Further, the citrate synthase from a variety of different animals seems remarkably constant in its properties " Restulation b~, Effectors A number of inhibitions of citrate synthase by metabolic intermediates have been reported and almost all of these interactions have been proposed to be regulatory for citrate synthase activity . We shall consider three groups of effectors, nucleotides, di- and tri-carboxylate anions, and inorganic ions . Wieland and Weiss (11) demonstrated an inhibition of citrate synthase by palmityl-CoA--the end product of fatty acid synthesis, a pathway that presumably is controlled by citrate concentration "

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This interaction was shown to be a non-specific detergent type of inhibition and probably does not have regulatory importance (12 ) . Hathaway and Atkinson (13) showed inhibition of citrate synthase by ATP--the final product of the Krebs cycle " They showed that ATP is a competitive inhibitor of acetyl CoA. Other nucleoside triphosphates were also effective inhibitors while ADP and AMP were much less effective as inhibitors " ATP is an inhibitor with a number of different citrate syntheses ; yeast (13 ), pig heart (1~+), beef heart (li+), beef liver (1~+), rat heart (9), rat liver (9), rat kidney (10), rat brain (10), lemon (15), trout liver (16), and B" subtilis (17 ) . The competition of ATP against oxalacetate is either noncompetitive or mused with the enzymes listed above " We have assayed the following enzymes and found them to be inhibited by ATP but we have not studied the complete kinetics of the system ; spinach, mango fruit, Azotobacter , and Aspergillus nidulans " Williamson and his coworkers using isolated rat heart mitochondria have presented evidence against ATP as a regulator (18,19) of citrate synthase activity " They have shown that succi nyl CoA (20) and propionyl CoA (21) are competitive inhibitors of acetyl CoA and have presented data that would implicate succinyl CoA as a physiological regulator of citrate synthase (20) " Recently, however,

Olson and Allgyer (22) have presented data

from an isolated liver mitochondria system which did not support the suggestion that succinyl CoA levels affected the rate of citrate synthesis "

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Srere (23) had shown earlier that propionyl CoA is an inhibitor for pig heart citrate synthase and recently Matsuoka and Srere (10,21+) using rat kidney and rat brain citrate synthase have shown that acetoacetyl CoA and NADH are also competitive inhibitors for acetyl CoA" This inhibition by acetoacetyl CoA allows the postulation of a regulatory mechanism which involves a positive feedback from ketone body formation" Formation of acetoacetyl CoA would inhibit citrate synthesis making more acetyl-CoA available for its own synthesis " Since the formation of acetoacetyl CoA depends on the square of the acetyl CoA concentration and the formation of citrate on the first power of acetyl CoA one would expect a buildup of acetoacetyl CoA at the expense of citrate " Experiments of Garland (25) with intact mitochondria show an exponential rise in ketones, tend to support such a mechanism " On the other hand, the high Ki (83 ~i) for acetoacetyl CoA and the very low cellular concentrations of acetoacetyl CoA makes this an unlikely control mechanism" Control of bacterial citrate syntheses is believed to be exerted in a different manner than that of the animal enzymes" Thus an inhibition by NADH has been observed with certain bacteri al enzymes which is reversed by AMP (26) " The claim has been made that ATP does not inhibit these bacterial citrate syntheses (27), and arguments based upon the different metabolic roles of the Krebs cycle in prokaryotes and eukaryotes have been used to support the NADH regulation of bacterial citrate synthase (28) as being physiologically significant " In addition, Weitzman and Ihumiore (28) have claimed that

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citrate synthase from pig heart and other so called "small" citrate syntheses are not inhibited by

NAD~fi

whereas "large"

citrate syntheses from certain microorganisms are inhibited. We have shown, however, that

NADH

(and

NADPH)

are inhibitors

of a small citrate synthase (29), and though the Ki's for these compounds are high, since neither

NAD

nor

NADP

are inhibitory,

the regulation by the redox couples is a plausible one " We have shown that the inhibitions by a-ketoglutarate and

NADH

are not

restricted to animal citrate syntheses but occur with plant citrate syntheses as well " Weitzman and Dunmore (27) have claimed that the differences of inhibitory interactions of bacterial and non-bacterial citrate syntheses fit a pattern that corresponds to the differing func tions of the Krebs cycle in the various organisms" Thus they and Wright et al . (30) showed that only citrate syntheses from the Enterobacteriacae of the gram negative bacteria were inhibited by a-ketogluterate" This observation was said by them to be related to the ability of the Enterobacteriacae to use the Krebs cycle as a pathway for the synthesis of a-lcetoglutarate " Recent studies on citrate synthase from Thiobacilli (31) and from halobacteria (32) do not fit this scheme . The citrate synthase from halobacteria, a gram negative organism, was a "small" enzyme, and was not sensitive to

NADH

inhibition "

We have shown that a-ketoglutarate does inhibit the citrate synthase of rat tissue " While it is true that its Ki against OAA is extremely high it should be remembered that free OAA concentration in mitochondria has been estimated to be very

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low (see below) . In addition the inhibition of E . coli citrate synthase by a-ketoglutarate is reversed (not seen at) by physiological concentrations of K+. The observations of Weitzman and Dunmore were made using a single set of assay conditions and inhibitor concentrations " Thus with the enzymes which have a low Km for oxalacetate inhibi tion by a-ketoglutarate will not be seen with 50 ~aM oxalacetate " It is, of course, still possible that these distinctions, in terms of relative inhibitory differences,

signals an important

clue as to the evolutionary development of the regulation of citrate synthase . Weitzman (33) has shown that NADH and a-ketoglutarate apparently affect the citrate synthase in toluenized E. coli cells" In a similar experiment Weitzman and Hewson (3~~) showed that ATP had no effect on the citrate synthase activity in toluenized yeast cells. This interesting technique may be useful in assessing the role of the various effectors on enzymes in situ but a number of questions must be answered (i " e . Were mitochondrial membranes made permeable? Why was the Km for acetyl CoA value so high?) before it can be accepted as the final word on the problem . Eggerer (35) was the first to note that the activity of pig heart citrate synthase was markedly affected by ionic strength . These observations were repeated by Srere (36), Wu and Yang (37) and Poulsen and Sarkissian (38) . Wu and Yang showed that increasing ionic strength increases the Km for acetyl CoA" The inhibition is competitive with acetyl CoA" Although there

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are differences in the effect of various salts in respect to their inhibitory power the main effect with animal enzymes seems to be an ionic strength effect . At low ionic strength, below 100 mM, citrate synthase activity decreases also . With bacterial enzymes specific ion effects have been seen . Weitzman (39) first noted that potassium ions reversed the effect of inhibitors of the E . cola citrate synthase . Work from our laboratories (~,0) showed that ions were necessary for the enzyme reaction, with K+, Na+ and NHS} + about equally effective while Li+ was 1/2 as effective. These ions not only were needed for activity but they strongly modified the action of other effectors of the E. cola enzyme ; K+ eliminates the NADH, palmityl CoA and a-ketoglutarate effects and enhances the ATP inhibition . K+ ion did not affect the molecular weight of E . cola citrate synthase as measured by gel filtration . On the other hand, small changes of the ultraviolet spectrum of the enzyme could be detec ted upon its addition to the E . cola enzyme (~}0 ) " Rowe and Weitzman have measured by electron microscopy small diameter increases of pure E. coli citrate caused by the addition of K+ (~}1 ) " Increasing ionic strength has been shown to be effective in protecting the pig heart enzyme against urea denaturation (23) " Wu and Yang (37) showed that increasing ionic strength changes the ORD spectrum of pig heart citrate synthase indicating a decrease in helicity . Divalent cations are inhibitory to both bacterial and animal citrate syntheses. One exception is the citrate (re)-synthase of C . acids urici (~~2) which has an absolute dependence on Mn+2

Controls of Citrate Synthase Activity

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2

vol . 15, No . 10

and to a smaller extent Zn+2 " Neither Mg+2 , Ca+2 or Fe +2

was effective as activators of the citrate (re)-synthase . Kosicki and Lee (~~.3,~+~}) have postulated that Mg+2 inhibits pig heart citrate synthase by binding to the polyphosphate chain of acetyl CoA " On the other hand, Wu and Yang (37) observed that increasing ionic strength whether with MgC1 2 or KSCN inhibits pig heart citrate synthase identically. In any case divalent cations,especially Mg

+2 ,

reverses the

ATP inhibition of pig heart (~+3) and rat liver (~+5) citrate synthesis" This is presumed to be äue to the fact that free ATP is the inhibitor while the MgATP complex is non inhibitory . Guynn et al . (~+6) have measured the equilibrium constant of the citrate synthase reaction under near physiological condi+2 ] = tions (38° ; pH 7 .0 ; I = 0"25 ; free [Mg 10 -3 ) and found it to be 2 .2~+ + 0" 1 x 10 6 (H2 0 = 1) . If free Mg was 1 x 10 6 " The effect of Mg

+2

+2 = 0, then this value

is a displacement of citrate

synthase reaction toward the direction of citrate synthesis. Substrates and Organization Lehrringer was the first to propose that citrate synthase was regulated by OAA concentration (i+7) . Later Randle and his coworkers (~+8) proposed that acetyl CoA concentration was limiting and regulates citrate synthase . Although no direct knowledge of the intramitochondrial concentrations of these two metabolites are available, there has been estimates of their free concentration based upon the assumption that most reactions in cells are near equilibrium" The equilibrium constant for the malate dehydrogenase is

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in the direction of oxalacetate formation so that at

malerte concentrations of 0" 3 mM and NADH/NAD ratios of 7, values determined from experiments with freeze clamped normal rat liver, (after making near equilibrium assumptions) free oxalacetate concentrations would be about ~~ x 10

-8

M. Similar values can be

calculated Pram data on total oxalacetate in tissue and the high concentrations of enzymes which bind it " Using similar methods with several other mitochondrial enzyme systems these workers estimated free acetyl CoA concentrations to be very low (50 ) " Since the Km for these substrates for the rat enzyme is about 5 ~.~M and Vmax is 1~~ ~mmoles citrate proäuced min/gm of liver then at such low substrate concentrations it is not likely that the citrate synthase could operate sufficiently fast to be part of the Rrebs cycle (~ = 1 ~aaole/min/gm) . To reconcile this inconsistency we can assume that either the calculation of free substrate concentration is in error or that the kinetic data on pure enzymes is not applicable to the enzyme in situ . We also can assume that these calculations of concentrations give an average value for wary microenvironmanta, and construct a new model that may explain the apparent inconsistency . This model includes the assumption that the Krebs cycle enzymes are arranged as a multienzyme system next to or on the inner surface of the inner mitochondrial membrane (3) so that locally high and low concentrations of intermediates exist as they are thought to do in conventional multienzyme systems. One requirement of such a system involving citrate synthesis

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is that oxalacetate concentration in the environment of citrate synthase be near its Km for that enzyme (5 x 10 -6 M) . However, if this is a multienzyme system and malate dehydrogenase is the source of this oxalacetate, then the microenvironment for MDH must contain at least that same concentration of OAA. I have pointed out earlier that the MDH reaction (Keq = 2 x 10 -5 ) cannot operate in the forward direction if [OAA] = 10 -6 M and NAD/NADH

m

7 . We are forced to conclude that the free redox

potential in the microenvironment of MDH is much different from this value . We can maintain the proper ratio by having the NADH transfer electrons immediately

to an acceptor in the inner

membrane and thus maintain a redox potential that permits MDH to operate . It is not particularly satisfying to proliferate special intracellular conditions such as the necessity to specify a different microenvironment for each enzyme . However, campartmenta tion phenomena have been demonstrated at a tissue, cell, organelle and multienzyme level and are believed to be a necessity for the orderly and controlled operation of cellular reactions . It is not too much of an extension of these ideas to believe that campartmentation exists even for a series of enzymes whose interactions are too weak to demonstrate by usual techniques " Model systems of immobilized enzymes have provided a means of constructing controlled microenvironments and have made it possible for multienzyme systems to determine the kinetic differ ences from a corresponding free enzyme in a single environment . Thus, when malate dehydrogenase and citrate synthase were immobi-

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lined on Sepharose (or trapped in acrylamide) and then the kinetics compared with the corresponding free enzymes (51), it was shown that at limiting malerte concentrations the immobilized enzyme system, in which the distance between enzymes were closer, operated at about twice the rate as a comparable system of free enzymes " Further, when lactate dehydrogenase was immobilized in the same system and pyruvate added as an electron acceptor, then rate enhancements of four fold over the free system were measured (51) " It is still not easy to dismiss all the 'kiear equilibrium" data used to calculate the free oxalacetate concentration " Perhaps it is still consistent in that it indicates that in spite of varying microenvironments in the same structural compartment of the cell the overall balance is maintained to keep the net thermodynamic state near equilibrium. ACIINOWLEDGE[~NTS This work was supported in part by a grant from the U.S .P .H .S . REFERENCES l.

P . A. SRERE, Current Topics in Cellular Regulation , p . 229

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P . A. SRERE, Energy Metabolism and the Regulation of Metabolic

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Controls of Citrate Synthese Activity

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Vol . 15, No . 10

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