302
`
`Biochimica et Biophysica Acta, 452 (1976) 302-309
`© Elsevier/North-Holland Biomedical Press
`
`BBA 67986
`
`MECHANISM OF THE INHIBITORY EFFECT OF GLYOXYLATE PLUS
`OXALOACETATE AND OXALOMALATE ON THE NADP-SPECIFIC
`ISOCITRATE DEHYDROGENASE
`
`OLE CHR. INGEBRETSEN
`
`Norsk Hydro's Institute for Cancer Research, The Norwegian Radium Hospital, Montebello,
`Oslo (Norway)
`
`(Received May 18th, 1976)
`
`Summary
`
`The effects of glyoxylate plus oxaloacetate and of oxalomalate on the
`NADP-linked
`isocitrate dehydrogenase
`(threo-Ds-isocitrate:NADP+ oxido(cid:173)
`reductase (decarboxylating, EC 1.1.1.42) from pig heart have been stud(cid:173)
`ied with steady state methods as well as with stopped flow technique. When
`equimolar mixtures of glyoxylate and oxaloacetate were premixed for dif(cid:173)
`ferent lengths of time prior to addition to the assay mixture, the extent of
`inhibition increased with the premixing time. The results indicated that the
`inhibition by glyoxylate plus oxaloacetate is caused by a compound formed
`in a reversible interaction between the two components.
`Glyoxylate plus oxaloacetate and oxalomalate affected the enzyme in at
`least three different ways. They inhibited the enzyme in a reaction competitive
`with regard to the substrate isocitrate. This inhibition needed a certain time to
`be fully expressed. The time lag could be eliminated by premixing of the
`enzyme and inhibitor with NADP plus metal ion. Secondly, if the enzyme is
`premixed with NADP plus metal ions, a time lag occurs before the reaction rate
`approaches a constant value after initiation of the reaction with isocitrate. The
`inhibitors were found to enhance this effect of NADP plus metal ions on the
`enzyme. Thirdly, it has previously been shown that the enzyme can be ac(cid:173)
`tivated by metal complexing agents. Glyoxylate plus oxaloacetate as well as
`oxalomalate are able to form complexes with metal ions and were found to
`cause an initial activation of the enzyme under certain assay conditions. The
`controversy regarding the mechanism of action of the above inhibitors on the
`enzyme is probably due to the fact that they affect the enzyme in several
`different ways.
`
`Introduction
`
`NADP-linked isocitrate dehydrogenases (threo-Ds-isocitrate:NADP+ oxido(cid:173)
`reductase ( decarboxylating), EC 1.1.1.42) from eukaryotic and prokaryotic
`
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`Page 1 of 8
`
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`

`303
`
`organisms are strongly inhibited by mixtures of glyoxylate and oxaloacetate
`[ 1-15]. Addition of one of the compounds alone has usually only a negligible
`effect on the enzyme activity. It is not established whether the inhibition is
`caused by a concerted action of the two compounds [3,4) or by a condensa(cid:173)
`tion product e.g. oxalomalate or -y-hydroxy o:-ketoglutaric acid [1,2,14,15).
`The mechanism of inhibition is not understood. Kinetic experiments have
`shown that the inhibition by glyoxylate plus oxaloacetate as well as by
`oxalomalate is competitive with regard to isocitrate [3,5,15). The fact that
`there is no simple structural analogy between isocitrate and the inhibitors [6],
`and the observation that the inhibition by glyoxylate plus oxaloacetate requires
`some minutes to be fully expressed [ 2, 7 ,13), suggest that the inhibition is not
`caused by simple competition for the active site. This conclusion is furthermore
`supported by the observation [ 8) that the enzyme can be modified by SH
`reagents in such a way that the inhibition by glyoxylate plus oxaloacetate is
`lost while the enzyme activity is unaffected.
`Isocitrate dehydrogenases need metal ions as cofactor, and Colman [16] has
`suggested that isocitrate complexed with metal ions is the "true" substrate
`for the enzyme and not isocitrate itself. Since glyoxylate and oxaloacetate as
`well as oxalomalate form complexes with metal ions in a similar way as the
`substrate isocitrate, the possibility exists that metal ions are involved in the in(cid:173)
`hibitory effect. We have recently found [ 17 ,18) that metal chelating agents
`may under certain conditions enhance the enzyme activity by a factor of more
`than 3 and remove the time lag found at low metal ion concentration before
`the absorption increases linearly with time. On the basis of this new
`information we have reinvestigated the mechanism of the inhibition by
`glyoxylate plus oxaloacetate and by oxalomalate by steady state kinetic
`methods as well as by stopped flow technique.
`
`Materials and Methods
`
`Materials. The NADP-dependent isocitrate dehydrogenase from pig heart,
`oxaloacetate and NADP were obtained from Boehringer, Mannheim GmbH,
`Germany. The enzyme was purified as described by Colman [19). DL-lsocitrate
`(trisodium salt), glyoxylate, oxalomalate and EGTA (ethyleneglycol-bis(t3-
`aminoethylether)) were purchased from Sigma Chem. Co., St. Louis, Mo.
`Assay of enzyme activity. The activity was measured from the increase in
`absorption at 340 nm upon reduction of NADP. All activity measurements
`were made at 25°C in 20 mM phosphate buffer, pH 8.0). The steady state
`kinetic studies were carried out with a Gilford Model 2400 recording spectro(cid:173)
`photometer. A Durrum stopped flow spectrophotometer (model D-100) was
`used in the presteady state kinetic experiments. Substrates and cofactor con(cid:173)
`centrations are given in the legends to the figures.
`Glyoxylate and oxaloacetate. The compounds were dissolved in 40 mM
`phosphate buffer (pH 8.0) immediately before the start of the experiments.
`Both compounds were found to be stable under these conditions. Where in(cid:173)
`dicated, the two compounds were premixed at equal concentrations and kept
`at room temperature for a certain period of time prior to the measurement
`of their inhibitory effect on the enzyme.
`
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`

`304
`
`Treatment of data. The changes in transmission in the stopped flow ex(cid:173)
`periments were displayed on a Tektronix storage oscilloscope. Photographs of
`the curves were taken with a Polaroid camera. The change in transmission was
`converted to absorbance and the data fitted to third order curves by the least
`square method [20]. The reaction rate was determined from the derivative of
`the curve. A Hewlett Packard calculator with an external X Y -plotter was
`used.
`
`Results
`
`The fact that some authors [2,7 ,13] find that it takes some minutes before
`the inhibition by glyoxylate and oxaloacetate is fully expressed, while others
`[ 4] do not observe any time lag, suggests that the lag may depend on the ex(cid:173)
`perimental conditions. Since it has been claimed [1,2,14,15] that the inhibi(cid:173)
`tion by glyoxylate plus oxaloacetate is caused by a compound formed by
`interaction of the two substances, the possibility was considered that the lag is
`affected by mixing of glyoxylate and oxaloacetate prior to addition to the
`assay mixture. Fig. lA shows that the lag time before the inhibition is fully
`expressed decreases with increasing mixing time of glyoxylate and oxalo(cid:173)
`acetate. Thus, when the two compounds had been premixed for 20 min prior
`to addition to the assay mixture, the time-absorbance curve becomes linear
`
`s
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`w
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`<(
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`<(
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`u z
`
`6
`4
`2
`REACTION TIME (MIN)
`
`60
`30
`90
`PREINCUBATION TIME (MIN)
`
`Fig. 1. Effect of premixing of g!yoxylate and oxaloacetate on the inhibition of isocitrate dehydrogcnase.
`(A) Increase in absorbance at 340 nm as a function of reaction time. G!yoxylate (10 mM) and oxalo(cid:173)
`acetate (10 mM) were preincubated at room temperature for the time indicated before addition to the
`assay mixture, and the reaction was initiated by addition of the enzyme. The final concentration in the
`assay mixture was 0.1 7 mM both for g!yoxy!ate and oxaloacetate (B). Enzyme activity in the presence of
`glyoxylate and oxaloacetate which had been subjected to different treatments prior to addition to the
`assay mixture. Solutions containing glyoxylate and oxaloacetate in a final concentration of 1 mM of each
`were made (1) by mixing of the two compounds (curve marked "mixing") or (2) by diluting a
`concentrated mixture containing 10 mM of each components with buffer, just prior to the initiation
`of the experiment. The concentrated mixture had been premixed for 30 min prior to the dilution. The
`inhibitory effect was determined as a function of time after mixing or dilution. The final concentration
`of g!yoxylate and oxaloacetate in the assay mixture was in all cases 8 nM. The assay mixture contained
`0.3 mM isocitrate, 0.05 mM NADP and 0.3 mM MgCl2.
`
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`Page 3 of 8
`
`

`

`305
`
`after approximately 3-4 min, while for premixing times of less than 1 min,
`more than 8 min were needed before the time absorption curve becomes linear,
`i.e. the inhibition is fully expressed. It should be stressed that in all cases the
`time-absorption curves eventually become linear. The results show, moreover,
`that the extent of inhibition increases with the mixing time of glyoxylate and
`oxaloacetate.
`Before studying in more detail the factors influencing the lag before the
`inhibition is fully expressed, further experiments on the effect of premixing
`on the extent of inhibition were performed. Fig. lB shows that the inhibitory
`effect of the equimolar mixture of glyoxylate and oxaloacetate increases
`strongly during the first 30 min after mixing, and subsequently approaches a
`constant value (see curve marked "mixing"). On the other hand, if a
`concentrated solution of glyoxylate and oxaloacetate (10 mM of each) mixed
`for 30 min (until maximum inhibitory effect was obtained) was diluted 10
`times with buffer, the inhibitory effect of this mixture decreases with time
`(curve marked "dilution"). In the experiments shown in Fig. lB the final
`concentration during the preincubation both in the mixing and in the dilution
`experiments was the same (1 mM of both glyoxylate and oxaloacetate). The
`activity was in all experiments calculated from the increase in absorption oc(cid:173)
`curring after the time absorption curve had become linear. It is apparent that
`the inhibitory effect of the two mixtures approaches the same value both in the
`mixing and in the dilution experiments after approximately 90 min. These
`results thus support the previous view that the inhibition by glyoxylate and
`oxaloacetate
`is caused by a compound formed from glyoxylate and
`oxaloacetate [1,2,14,15]. Moreover, the inhibitory compound is formed in a
`reversible reaction.
`Further information on the factors influencing the time lag before the
`inhibition by glyoxylate and oxaloacetate is fully expressed was sought in
`experiments using stopped flow spectroscopy. The experiments shown in Fig. 2
`have been carried out both with glyoxylate plus oxaloacetate as well as with
`oxalomalate. However, since the effect of glyoxylate and oxaloacetate depends
`strongly on the premixing conditions the experiments are more difficult to
`perform, and the data obtained with oxalomalate are presented. It should be
`that the results with mixtures of glyoxylate and oxaloacetate
`stressed
`corresponds closely to those shown for oxalomalate.
`A small lag time is observed in the stopped flow experiments before the
`absorption started to increase linearly with time. This lag [ 17] is considerably
`greater when the enzyme is premixed with NADP and Mg2+ (Fig. 2B) than
`when premixed with isocitrate and Mg2
`+ (Fig. 2A) prior to initiation of the
`reaction. Interestingly, when oxalomalate was mixed with the enzyme at the
`same time as the reaction was started, the lag time disappeared. Thus, under
`these conditions oxalomalate activated the enzyme immediately after initia(cid:173)
`tion of the reaction. The slope of the absorption curve obtained in the pres(cid:173)
`ence of oxalomalate levelled off after a short time, and the inhibition was
`fully expressed after approximately one min. On the other hand, if the en(cid:173)
`zyme was premixed with NADP and Mg2
`+ together with oxalomalate the
`inhibition was fully expressed when the reaction was initiated by addition of
`isocitrate (Fig. 2B). With all other combinations of substrate, co-factor and
`
`Rigel Exhibit 1035
`Page 4 of 8
`
`

`

`--
`Jo.3 E•NADP•MgCl 2 ~
`.,
`
`IC
`
`A
`
`B
`
`Control
`
`306
`
`--
`Jo.3 E•IC•MgCl 2 =:)--
`.,
`
`NAOP
`
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`
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`
`100
`150
`50
`REACTION TIME (s)
`
`150
`100
`50
`REACTION TIME (s)
`
`Fig. 2. Effect of different premixing conditions on the inhibition by oxalomalate. (A) Increase in ab(cid:173)
`sorption as a function of time after initiation of the reaction by addition of N ADP. The enzyme was pre(cid:173)
`incubated with isocitrate and MgCl2. Oxalomalate was present as indicated. (B) Increase in absorption
`as a function of time after initiation of the reaction by addition of isocitrate. The enzyme was pre(cid:173)
`incubated with NADP and MgCl2, Oxalomalate was present as indicated. The experiments were carried
`out with stopped flow spectrophotometry. The subscript to oxalomalate in the text on the figures
`indicate whether the inhibitor was present together with the enzyme (E) or with the substrate used to
`initiate the reaction (S). The final concentrations were: 0.9 mM isocitrate, 0.05 mM NADP and 0.03 mM
`MgCl2 and 2 mM oxalomalate where indicated.
`
`co-enzyme it was found that a lag time was needed before the inhibition was
`fully expressed independently of whether oxalomalate was premixed with the
`enzyme or not. From these experiments it can be concluded that the inhibitor
`reacts with the enzyme in a relatively slow reaction and that this reaction
`requires both NADP and metal ion.
`One possible interpretation of the above results is that the inhibitor is
`converted to a second, more potent inhibitor by the enzyme in a reaction
`requiring NADP plus metal ions. In order to test this hypothesis, additional
`enzyme was added to an assay mixture after the inhibition had been fully
`in
`this
`(Fig. 3A). Glyoxylate plus oxaloacetate were used
`expressed
`experiment. It is apparent that a new lag occurs after addition of more enzyme
`before the inhibition is fully expressed again. Moreover, the results show in
`agreement with the data in Fig. 2B, that the lag disappears if the inhibitors are
`preincubated with enzyme, NADP and metal ions prior to initiation of the
`reaction with isocitrate. Preincubation of the inhibitors with enzyme, isocitrate
`and metal ions had no effect on the lag time.
`Since glyoxylate plus oxaloacetate as well as oxalomalate can form com(cid:173)
`plexes with metal ions, the effect of the metal concentration on the inhibition
`was studied. Fig. 3B shows that the inhibition increases with increasing Mg2
`+
`concentration. These results thus show that the inhibition cannot be caused
`by a competition with isocitrate for the metal ions. Ruffo et al. [2] have
`+ on the inhibition. They found
`previously studied the effect of Mn 2+ and Mg2
`in contrast to our results that the inhibition patterns with Mg2
`+ were different
`for oxalomalate and mixtures of glyoxylate plus oxaloacetate.
`
`Rigel Exhibit 1035
`Page 5 of 8
`
`

`

`307
`
`Glyoxylate •
`oxaloacetate
`
`;-' 10 B
`0
`
`~
`0
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`w
`~ 0 04
`<(
`CD g 003
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`
`2
`4
`6
`REACTION TIME (MIN)
`
`0.2
`
`0.4
`
`06
`
`Mg Cl 2 CONC. (mM)
`Fig, 3 . (A) Increase in absorbance as a function of reaction time in the absence and presence of glyoxylate
`plus oxaloacetate . Aliquots of glyoxylate plus oxaloacetate were added to the assay mixture 15 s prior
`to the initiation of the reaction by addition of enzyme, isocitrate or NADP. When the reaction was
`initiated b y the enzyme, an equal amount of additional enzyme was added after 4 min. The concentration
`of MgCl2 was 0.3 mM . (B) Enzyme activity as a function of MgCl2 concentration. The activity was
`measured in the absence of any inhibitors as well as in the presence of glyoxylate plus oxaloacetate (4
`nm) of each, and oxalomalate (0.25 mM). The g)yoxylate plus oxaloacetate had in all experiments been
`premixed in a concentration of 10 mM for 30 min . The assay mixture contained 0.3 mM isocitrate and
`0.05 mM NADP .
`
`We have previously found [17,18] that metal chelating agents remove the
`time lag and increases the enzyme activity. In Fig. 4 the effect of ethylene(cid:173)
`glycol-bis(/3-aminoethylether) N,N' -tetraacetic acid (EGT A) on the inhibition by
`different concentrations of oxalomalate was studied. Oxalomalate was in all
`experiments preincubated with the enzyme together with NADP and metal
`ions. The results in the absence of the activator, EGTA, is shown in Fig. 4A.
`The lag increases with increasing concentrations of oxalomalate. In fact, there
`is a linear relationship between the lag time before the absorption increases
`linearly with time, and the concentration of oxalomalate (insert). We have
`previously obtained evidence [ 17] that NADP plus metal ions transform the
`enzyme to a nonactive state. The lag time observed is in part explained on
`this basis. Apparently, oxalomalate enhances the effect of NADP and Mg2
`•
`in transforming the enzyme. Consequently, the time needed for isocitrate to
`activate
`the enzyme will
`increase with
`increasing concentrations of
`oxalomalate.
`EGTA prevents the induction of the non-active state by NADP plus metal
`ions [18]. In the presence of EGTA (Fig. 4B) no lag is observed (compare
`curves marked No EGTA and Control), and
`the
`inhibitory effect of
`oxalomalate is fully expressed from the start of the reaction. Interestingly,
`oxalomalate inhibited the enzyme to a considerably larger extent in the pres(cid:173)
`ence of EGT A (insert, data taken both from panel A and B). Thus, it is
`apparent that 50% inhibition in the absence of EGT A required 2-3 times
`higher oxalomalate concentration than in the presence of EGT A.
`
`Rigel Exhibit 1035
`Page 6 of 8
`
`

`

`E•NAOP•MgCt,• OM=>-
`
`10
`
`•
`
`0.2 0.L
`[OM] (IT-Ml
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`
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`
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`
`100
`150
`50
`REACTION TIME (s)
`
`150
`50
`100
`REACTION TIME (s)
`
`Fig. 4 . The inhibitory effect of oxalomalate. The increase in absorbance as a function of time after mixing
`was determined in the absence (A) and in the presence of EGTA (B). The enzyme was in all experiments
`preincubated with NADP and MgClz. Oxalomalate and EGTA were present together with the enzyme as
`indicated . The reaction was initiated by the addition of isocitratc. The insert in Panel A shows the lag
`time as a function of oxalomalate concentration. The lag time was determined by extrapolation of the
`curve to zero change in absorption as indicated. The insert in Panel B shows the inhibition as a function of
`the oxalomalate concentration in the absence and presence of EGTA (data taken both from Panel A and
`B). The experiments were carried out with the stopped flow spectrophotometer. The final concentrations
`were: 0.3 mM isocitrate, 0.05 mM NADP, 0.03 mM MgClz and 0.03 mM EGTA where indicated. Since
`EGTA activates the enzyme, Jess enzyme protein was used in the experiments shown in Panel B than in
`those in Panel A .
`
`Discussion
`
`The present results indicate that the inhibition of the NADP-dependent
`isocitrate dehydrogenase by glyoxylate plus oxaloacetate and by oxalomalate
`are caused by interaction of the inhibitors with a site different from the
`binding site of the substrate, and that the allosteric inhibition is mediated
`through a relatively slow change in the conformation of the protein. The
`results support the view [ 1,2,14,15] that the inhibition by glyoxylate plus
`oxaloacetate is caused by a complex or condensation product formed between
`the two substances. In all experiments it was found that oxalomalate which is
`formed upon mixing of glyoxylate and oxaloacetate (1,14,15] showed the same
`inhibition pattern as mixtures of glyoxylate plus oxaloacetate. It is not possible
`from the present experiments to decide whether the condensation product
`responsible for the inhibition upon mixing of glyoxylate and oxaloacetate is
`oxalomalate or another compound.
`Previous authors [3,5,15] have found that glyoxylate plus oxaloacetate as
`well as oxalomalate inhibit the enzyme in a competitive manner with regard to
`isocitrate, and in a noncompetitive manner with regard to NADP. Our results
`confirm this (data not presented). On this basis it has been claimed that the
`inhibition is non-allosteric. The present results do not support this conclusion.
`Thus, the inhibition needs a considerable time to be fully expressed unless the
`inhibitors are preincubated with the enzyme together with NADP plus metal
`
`Rigel Exhibit 1035
`Page 7 of 8
`
`

`

`309
`
`ions. Secondly, we have previously shown [18] that citrate which competes
`with isocitrate for the active site inhibits the enzyme to the same extent in(cid:173)
`dependently of whether the activator EGTA is present or not. EGTA itself
`has only a slight effect on Km for isocitrate [18]. If oxalomalate inhibited the
`enzyme by competing for the active site the same inhibition pattern would be
`expected as found for citrate. However, the present results show that
`oxalomalate inhibits the enzyme considerably more efficiently when the
`enzyme is activated by the presence of EGT A than in the absence of EGT A.
`The results show that glyoxylate plus oxaloacetate and oxalomalate affects
`the enzyme in several ways. As discussed above they inhibit the enzyme in a
`competitive manner with regard to isocitrate. Secondly, the inhibitors increases
`the lag time found when the enzyme is preincubated with NADP plus metal
`ions prior to initiation of the reaction with isocitrate. We have previously found
`that NADP and metal ions convert the enzyme to a non-active state [17] and
`apparently the inhibitors enhance this effect. Thirdly, due to the ability of the
`inhibitors to form complexes with metal ions they are able to activate the
`enzyme in a similar way as previously shown for metal chelating agents [18].
`The fact that the enzyme is affected in several different ways by the inhibitors
`is probably the reason for the controversy concerning the mechanism of in(cid:173)
`hibition by glyoxylate plus oxaloacetate.
`
`Acknowledgements
`
`This work was supported by The Norwegian Research Council for Science
`and the Humanities. The author is indepted to Dr. T. Sanner for helpful dis(cid:173)
`cussions. The able technical assistance of Mrs. Astri Nordahl and Mrs.
`Margareth Skogland is gratefully acknowledged.
`
`References
`
`1 Ruffo, A., Testa, E., Adinolifi, A., Pelizza, G . and Moratti, R. (1967) Biochem. J. 103, 19-23
`2 Ruffo, A., Moratti, R ., Montani, A. and Melzi D'Eril, G.L. (1974) Ital. J. Biochem. 23, 357-370
`3 Shilo, I. and Ozaki, H. (1968) J. Biochem. Tokyo 64, 45-53
`4 Marr, J .J. and Weber, M.M. (1969) J. Biol. Chem. 244, 5709-5712
`5 Marr, J.J . and Weber, M.M. (1969) Biochem. Biophys. Res . Commun. 35, 12-19
`6 Marr, J.J. and Weber, M.M. (1971) Biochem. Biophys. Res. Commun. 45, 1019-1024
`7 Charles, A .M. (1970) Can. J. Biochem. 48, 95-103
`8 Little, C. and Holland, P. (1972) Can. J. Biochem. 50, 1109-1113
`9 Glaeser, H. and Schlegel, H.G. (1972) Arch. Microbiol. 86, 327-337
`10 Ramaley, R.F. and Hudock, M.O. (1973) Biochim. Biophys. Acta 315, 22-36
`11 Self, C .H ., Parker, M.G . and Weitzman, P.D.J. (1973) Biochem. J. 132, 215-221
`12 Kleber, H.P. (1975) Z . Alig . Mikrobiol. 15, 431-435
`13 Ingebretsen, O.C. (1975) J . Bacteriol. 124, 65-72
`14 Payes, B. and Laties, G.G. (1963) Biochem. Biophys. Res. Commun. 10, 460-466
`15 Adinolfi, A., Moratti, R ., Olezza, S. and Ruffo, A. (1969) Biochem. J. 114, 513-518
`16 Colman, R.F. (1972) J. Biol. Chem. 247, 215-223
`17 Sanner, T . and Ingebretsen, O.C. (1976) Arch. Biochem. Biophys. 172, 59---63
`18 Ingebretsen, O.C. and Sanner, T. Arch. Biochem. Biophys., in the press.
`19 Colman , R.F . (1968) J. Biol. Chem. 243, 2454-2464
`20 Sanner, T. (1971) Biochim. Biophys. Acta 250, 297-305
`
`Rigel Exhibit 1035
`Page 8 of 8
`
`