TRANSHYDROGENASE REGULATION 11
Biochemistry 5, 365-38 5 . Louie, D. D., and Kaplan, N. 0. (1970), J . Biol. Chem. 245, 5691 -5698. Louie, D. D., Kaplan, N . O., and McLean, J. D. (1972), J . Mol. Biol. 70, 65 1-664. Monod, J., Wyman, J., and Changeux, J . P. (1965), J . Mol. Biol. 12, 88-118. P-L Biochemicals, Inc., Circular OR- 18 (1 965), Ultraviolet Absorption Spectra of Pyridine Nucleotide Coenzymes and Coenzyme Analogs. Rydstrom, J., Hoek, J. B., and Hojeberg, B. (1973), Biochem. Biophys. Res. Commun. 52, 421-429. Walter, P., and Rubin, B. (1966), Biochem. Prep. 10, 166170. Webb, J. L. (1963), in Enzymes and Metabolic Inhibitors, Vol. 2, New York, N.Y., Academic Press, 439, 446. Widmer, F., and Kaplan, N. 0. (1976), Biochemistry 15, (following paper in this issue).
Colowick, S. P., Kaplan, N. O., Neufeld, E. F., and Ciotti, M. M. (1952), J . Biol. Chem. 195, 95-105. Dieter, H., Koberstein, R., and Sund, H. (1974), FEBS Lett. 47, 90-93. Ginsburg, A., and Mehler, A. H. (1966), Fed. Proc., Fed. Am. SOC.Exp. Biol. 25, 407. Goldbeter, A. (1974), J . Mol. Biol. 90, 185-190. Hojeberg, B., and Rydstrom, J. (1976), Znt. Congr. Biochem., IOth, 07-4-117. Kaplan, N. 0. (1957), Methods Enzymol. 3, 873-876. Kaplan, N. 0. (1972), Haroey Lect. 66, 105-133. Kaplan, N. O., Colowick, S. P., and Neufeld, E. F. (1952), J . Biol. Chem. 195, 107-1 19. Kaplan, N. O., Colowick, S. P., Neufeld, E. F., and Ciotti, M. M. (1953), J . Biol. Chem. 205, 17-29. Kirtley, M. E., and Koshland, D. E., Jr. (1967), J. Biol. Chem. 242, 4192-4205. Koshland, D. E., Jr., Nemethy, G., and Filmer, D. (1966),
Regulatory Properties of the Pyridine Nucleotide Transhydrogenase from Pseudomonas aeruginosa. Active Enzyme Ultracentrifugation Studies? FranGois Widmert.8 and Nathan 0. Kaplan*
ABSTRACT: Active enzyme ultracentrifugation studies of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa (EC 1.6.1.1.) show that the enzymatic reaction is catalyzed by a molecular species characterized by an ~ 2 0 value of about 34 S, whatever the reduced substrate may be (tri- or diphosphopyridine nucleotide). The filamentous aggregated form of the enzyme ( ~ 2 0 = , ~ 121 S and higher), identified by previous investigations (Cohen, P. T., and Kaplan,
w e have reported, in the preceding paper of this issue (Widmer and Kaplan, 1976), that the MWC allosteric model (Monod et al., 1965) might be used as a framework to explore the regulatory characteristics of the enzyme transhydrogenase from Pseudomonas aeruginosa. The R state is favored by TPNH,’ 2’-AMP, and several other 2’-phosphate nucleotides, whereas TPN+ and inorganic phosphate show more affinity for the T state. On the other hand, the structure of PATH, as elucidated by ultracentrifugation (Cohen, 1967; Cohen and Kaplan, 1970a) and electronmicroscopy (Louie et a]., 1972), might prompt one to assume that association-dissociation + From the Department of Chemistry, University of California, San Diego, La Jolla, California 92093. Receiued April 6, 1976. This work was supported by grants from the American Cancer Society (BC-60-P) and from the National Institutes of Health (USPHS) (CA 11683-0s). Postdoctoral fellow of the Swiss National Science Foundation. 5 Present address: NestlC Products Technical Assistance Co. Ltd., Research Department, Biochemistry Section, P.O. Box 88, CH-I 8 14 La Tour-de-Peilz, Switzerland. I Abbreviation: PATH, Pseudomonas aeruginosa pyridine nucleotide transhydrogenase; for other abbreviations, see footnote 1 of the preceding paper in this issue (Widmer and Kaplan, 1976).
*
,
N. 0. (1970), J . Biol. Chem. 245, 2825-2836; Louie, D. D., Kaplan, N. O., and Mc Lean, J. D. (1972), J . Mol. Biol. 70, 65 1 -664), appears, therefore, to be an inactive species. The ~ physiological implicationsof the enzyme are discussed. Several lines of evidence lead to the conclusion that the transhydrogenase might act as an essential link between carbohydrate catabolism and the respiratory chain.
reactions could characterize the catalytic mechanism of the enzyme. Such a regulatory mechanism would not be in agreement with the fairly general rule that allosteric enzymes have a fixed number of protomers, which is independent of any allosteric transitions which might occur. The main component of the sedimentation pattern of PATH in its native form is characterized by an ~ 2 0value , ~ of 121 S, whereas aggregated material, not actually in solution, sediments with an even higher speed (Cohen and Kaplan, 1970a). These two components should correspond to the rodlike shaped polydisperse structure seen on electronmicrographs of the native enzyme (Louie et al., 1972). On the other hand, Cohen and Kaplan (1970a) found that in the presence of 1 mM 2’AMP or 1 mM TPN+ the sedimentation pattern is homogeneous, and characterized by a single component with an ~ 2 0 , of 33.8 S. This component has been assumed by Louie et al. ( 1972) to correspond to the uniform population of cylindrical particles (900 000 dalton) seen on electronmicrographs after addition of 1 mM 2’-AMP. A very small amount of such units are already seen on electronmicrographs of the native structure, and should correspond to the component with an ~ 2 0of, ~ BIOCHEMISTRY,
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Actiue Enzyme Ultracentrifugation. The experiments were carried out according to the method of Kemper and Everse (1973). using a Spinco Model E analytical centrifuge equipped with a photoelectric scanning system. All the reagents for the assays were prepared in 0.1 M Tris buffer (pH 7.5) containing IO mM 2-mercaptoethanol. The PATH dilutions were prepared in 0.01 M Tris buffer (pH 7.5). also containing IO m M 2-mercaptcethanol. Ten microliters of these dilutions were used for each experiment a t the concentrations indicated in the text.
. . . . . .,:
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about 34 S, which can be observed in a low concentration on the heterogeneous sedimentation pattern of native PATH (Cohen and Kaplan, 197ba). Three different components have thus been identified on this pattern; they have been called components I, 11, and III (I: ~ 2 0 =. 33.8 ~ S; 11: s~o,,, = 121 S; 111: aggregated material). The active enzyme ultracentrifugation technique first developed by R. Cohen (1963) and modified by Kemper and Everse ( I 973) allows for the determination of the value of an enzyme in its catalytically active form; this determination is done under kinetic assay conditions. To date, the following reactions of PATH have been studied with this technique: 2'-AMP-activated DPNH-(TN)DPN+ reaction, 2'-AMPactivated TPNH-(TN)DPN+ reaction, and TPNH-(TN)DPN+ reaction (Louie et al., 1972). The last named reaction was characterized by an 320.wvalue of 1IO S, the other ones by an s20.wvalue between 28 S and 30 S. It was therefore possible to correlate the activation by 2'-AMP with the disaggregation process. As for the third reaction, the ~ 2 0 of . ~1 IO S was issumed to indicate that the rodlike structure is the active species for the given reaction (Louie et al., 1972). This assumption, we now believe, was not correct, mainly because no T P N H regenerating system was used. Consequently, the concentrations of the allosteric ligands T P N H and TPN+ were not kept constant. For such a situation, it is likely that the ultracentrifugation pattern was affected by the continuous shift of the allosteric equilibrium throughout the experiment. Furthermore, the ~ 2 0 value . ~ yielded by an active enzyme ultracentrifugation can be an apparent value only (as for usual ultracentrifugation techniques) if the enzyme is characterized by an association-dissociation equilibrium. All the available evidence suggests that PATH is such a system. The main goal of the present report was to ascertain whether the filamentous aggregated form of PATH might really represent an active species. It appears from our results that disaggregation into particles of 900 000 molecular weight is a prerequisite to enzymatic activity. Materials and Methods Chemicals and Enzymes. DL-Sodium isocitrate was purchased from Calbiochem. All other reagents, as well as PATH, were obtained as indicaied in the preceding paper of this issue (Widmer and Kaplan, 1976). TPN+-specific isocitrate dehydrogenase from pig heart was purified by Mr. F. E. Stolzenbach according to the procedure of Ochoa (1955).
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DPNH-(TN)DPN+Reaction. This reaction proceeds a t a very low rate in the absence of 2'-AMP, but is not negligible, particularly when the solutions are buffered with Tris. The active enzyme ultracentrifugation of this reaction was carried out with five different PATH concentrations, between 0.02 and 0.0035 enzyme units in the 10 pl initiating the reaction, and with substrate concentrations of 0.1 mM. A heterogeneous pattern was always obtained and on each scan three different reg.ons could be identified, as exemplified by Figure 1. Part I: a well defined boundary is moving down with an ~ ~ valueof 33.7S.ThesemilogplotoflogXvs. time(Xbeingthe distance between the center of rotation and the boundary) is straight for about 40 min, then slightly bends downward. This s 2 0 . value ~ of 33.7 S fits quite well with the ~ 2 0value . ~ of 33.8 S characterizing the TPN+ or 2'-AMP-induced structure of PATH (Cohen and Kaplan, 1970a). Part 11: the constant absorbance increase in this part should be due to a uniformly distributed active enzyme species. Part 111: a significant activity can be seen a t the bottom of the cell as soon as the run starts, and the absorbance increase appears as if due to a diffusion process. When the concentration of enzyme is raised, the characteristics of parts I1 and III become more conspicuous but, in this case, all three parts are somewhat overlapping (compare scans A and B un Figure 1). For a given enzyme concentration, the absorbance values seen in parts I1 and 111 are increasing with time. The heterogeneous pattern just described can be reconciled with the very first ultracentrifuge runs of native PATH by Cohen and Kaplan (1970a). because parts I, 11, and III seen on our scans can be explained by components I, 11, and 111 identified by these authors under usual ultracentrifugation conditions (high enzyme concentration, absence of any substrate or effector). In both cases, the heterogeneity of the ultracentrifuge patterns can be interpreted as the indication of a very slow interaction (if at all) among different enzyme species. Therefore, the similarity between the native enzyme pattern and the active enzyme pattern for the DPNH(TN)DPN+ reaction suggests that, when PATH is layered as a thin film on the substrate solutions in the ultracentrifuge cell, the nonuniform population remains characterized by the three components described for the native enzyme by Cohen and Kaplan (l970a). This may be related to the fact that the substrates involved in the reaction are unable to alter the allosteric equilibrium (Widmer and Kaplan, 1976). A consistent explanation of our results is possible if one assumes that only component I is significantly active for the DPNH-(TN)DPN+ reaction. The straight log Xvs. time plot characterizing the boundary of part I, which contains most of the enzymatic xtivity, is indeed explained by the sedimentation of a homogeneous active enzyme species with an S Z O . of ~ 33.7 S. The slight loss of activity detected after 40 min, and
0
,
~
TRANSHYDROGENASE REGULATION I1
elicited by the downward bending of the semilog plot, might be accounted for by a slow reassociation p r o x s (in compliance with the interconversion equilibrium), with a resulting faster sedimentation of the created aggregates. The assumption of a slow interconversion rate can also explain the features of parts 11 and Ill. The diffusion-like absorbance increase at the bottom of the cell (part H I ) , which can be seen even in the very early stages of the runs, definitely recalls to mind the very fast sedimentation of component 111 in the ultracentrifugation of native PATH (Cohen and Kaplan, 1970a). If the different species present in the heterogeneous native population (not affected b) DPNH or ( T U ) D k + )arechxdc&ed by interconver$ion equilibria. component 111 must disaggregate to some extent, in compliance with the equilibrium constants. Therefore. it is our view that the enzymatic activity seen in part I l l is due to 3 4 s particles created by the pariial disaggregation of component 111. A similar line of argument leads to assume that the enzymatic activity seen in part I1 might be due to 3 4 s particles resulting from the partial disaggregation of an enzyme species probably corresponding to the 121s component. However, it is not yet fully explained why this activity seems to be uniformly distributed in part I1 (this distribution could be due to a pression and/or concentration effect). The assumption that the 3 4 s particles are the only significantly active species is substantiated by the fact that most of the activity is found in part I, where this species is found as a homogeneous population, while making up only a minute percentage of the total protein in the ultracentrifuge cell (Cohen and Kaplan, 1970a: Louie et al., 1972). TPNH-( TN)DPN+ Reaction with Regenerating System (Saturating Amount of T P N H ) . The substrate T P N H is assumed to shift the R-T allosteric equilibrium towards the R state, whereas the product TPN+ favors the T state (Widmer and Kaplan, 1976). In such a situation, it is obvious that any active enzyme ultracentrifugation pattern will be complicated by the continuously varying TPNH and TPN+ concentrations if no T P N H regenerating system is used. It is therefore advisable to include such a system in the contents of the ultracentrifuge cells. In so doing, the concentrations of allosteric ligands will be kept constant. The regenerating system we used for our experiments was the TPN+-specific isocitrate dehydrogenase system, as described by Cohen and Kaplan (1970b). For PATH, the experimental conditions consisted of 0.3 mM TPNH, 0.1 mM (TN)DPN+, and 0.001 enzyme unit. A homogeneous pattern was obtained (Figure 2A) with a single symmetrical boundary Characterized by an ~ 2 0 of. ~35.0 S. No loss of activity was encountered during the runs and no activity whatsoever could be detected in the rest of the cell. The presence of 0.3 mM T P N H can, therefore, be assumed to induce a homogeneous enzyme population; the ~ 2 0value . ~ is in agreement with that of the 900 000 molecular-weight species. The homogeneous pattern indicates that the structural alteration induced by T P N H is very rapid, compared to the slow interconversion rates assumed in the absence of allosteric ligands (see preceding paragraph). I t has been reporled that 2':AMPactivation can no longer be seen for theTPNH-(TNIDPN' reaction when theTPKH . , concentration reaches 0.3 mM (Cohen and Kaplan, 1970a). This fact can be reconciled with our present finding that at this concentration the allosteric ligand T P N H induces the formation of a homogeneous population similar to the 2'-AMPinduced population. TPNH-( TN)DPN+ Reaction without Regenerating System (Saturating Amount of TPNH). When the regenerating system is omitted, the pattern is heterogeneous (Figure 2B)
... .. ..._ . ... . ... ....... ... . . :!
. ....
FIGUKt. 2 Scans of Ihc T P N H - ( T N ) D P N + racliun. about 40 nun after the start of the runs (24 630 rpm: 0.001 P A T H units). ( A ) with T P N H regenerating system: (B) without TPNH regenerating system. Monitoring: as far Figure I
and strongly resembles the heterogeneous patterns of the DPNH-(TN)DPN+ reaction (Figure I). However, there are two major differences. First the log X vs. time plot for the boundary corresponding to part I bends downward from the outset (the initial slope of this plot yields an ~ 2 0of. about ~ 35 S). Secondly, the increase with time of the absorbance seen in part I I is not uniform throughout the ultracentrifuge run. A fast moving "shoulder" is elicited towards the end of the run. which would inevitably mask the 35 S boundary (part I), should PATH be used a t a higher concentration. This complicated ultracentrifugation pattern in the absence of regenerating system is certainly due to the fact that the concentrations of TPNH and TPN+ are not kept constant throughout the experiment. TPNH-( TN)DPN+ Reaction with Regenerating System (Subsaturating Amount of TPNH). As a complement to the two preceding studies, the TPNH-(TN)DPN+ reaction with TPNH regenerating system was also studied at a relatively low TPNH concentration ( I O ,LMTPNH; (TN)DPN+ and PATH were maintained at their previous concentrations). The significant decrease of T P N H concentration has the expected consequence that a homogeneous enzyme population is no longer induced, since the given concentration is far from being saturating. The scans correspond somewhat to the pattern illustrated in Figure 2B. However, there is a conspicuous difference: the main component, which shows an ~ 2 0of. 33.3 ~ S , is characterized by a constant activity for at least 40 min, i.e., the log X vs. time plot is a straight line for this period of time (the subsequent downward bending is hardly seen). It is therefore possible to conclude that. in this case, the active species also corresponds to the low ~ 2 0 species. . ~ The occurrence of heterogeneous material in the rest of the cell is unavoidable, since the concentration of the allosteric ligand T P N H is well under the saturating level. The following statements summarize the results of the UIlracentrilugation \tudies: (I)The native form of PATH is a heteroeeneous ~onulation.The interconversion rdteb areslow. compared to those of the centrifugal separation of the components. (2) With the DPNH-(TN)DPN+ reaction, PATH retains its native form ( I 2 1 s component and aggregated material). The elicited transhydrogenase activity is accounted for by the 3 4 s particles already present in the nonuniform native population and by similar particles appearing during centrifugal separation throughout the cell by the slow breaking down of the large aggregates. (3) With the TPNH-(TN)DPN+
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P
~
FADH;!
P Glyox late N
DPN’
ADP -
FIGURE 3: Metabolic significance of PATH (WDH: Warburg-Dickens-Horecker: ED: Entner-Doudoroff).
reaction, a rapid structural alteration of PATH occurs as soon as the enzyme is layered on the substrates solutions. With a constant saturating concentration of TPNH, the entire enzyme population corresponds to the 34s species. If the concentration of T P N H is not saturating, the 3 4 s induction is not complete and some larger entities are observed. (4) The dilution to assay conditions does not promote in itself the disaggregation of the rodlike polydisperse structure characterizing the native isolated form of PATH. Discussion Cohen and Kaplan (1970a), as well as Louie et al. (1972), have shown that the polydisperse structure of PATH is a characteristic of the native isolated form of the enzyme. It appears from the present ultracentrifugation results that most, if not all, of the transhydrogenase activity is associated with the low-molecular form of the enzyme (34s species). This was found to be the case with whatever reduced substrate was used (DPNH or TPNH), which means that the filamentous aggregates have little or no enzymatic activity. The present work also shows that the association-dissociation equilibrium characterizing the native PATH is not significantly affected by the presence of DPNH and (TN)DPN+, whereas T P N H is able to induce a homogeneous 3 4 s population. The disaggregation of the inactive filamentous aggregates is therefore related to the presence of a ligand favoring the hypothetical R form of the enzyme, since TPNH-unlike DPNH and (TN)DPN+-has been recognized as an allosteric ligand (Widmer and Kaplan, 1976). In terms of the MWC model, the inactive aggregates would, therefore, somehow correspond to the nonfunctional T form, which might be prone to spontaneous aggregation in the absence of any allosteric ligands. This does not correspond to any basic assumption or prediction of the MWC model, which we have used as a framework to explain the catalytic properties of the transhydrogenase (Widmer and Kaplan, 1976). One has to keep in mind, however, that the model offers only an oversimplified first approximation of real systems, and that the “state” of an allosteric protein may not in fact be exactly the same whether it is actually bound or unbound to as stabilizing ligand (Monod et al., 1965). It has been shown that the low-molecular-weight form of PATH (34s species) possesses 20-24 FAD’s, presumably corresponding to the same number of protomers (Louie et al., 1972). Such a large size is not a priori incompatible with the symmetry requirement of the MWC model, since it has been shown that this requirement can be fulfilled for oligomers containing as many as 24 or 60 protomers (Hanson, 1966; Haschemeyer and de Harven, 1974). For PATH, association-dissociation processes would have
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little importance in vivo, since the enzyme is always in the presence of T P N H and/or T P N + , which stabilize its 34s forms. It is not possible to rule out that the association-dissociation processes might possibly be the result rather than the H ~ prime causes of the allosteric mechanisms of PATH. In this connection, it is worth quoting that experiments with immobilized beef liver glutamate dehydrogenase (EC 1.4.1.3) have recently shown that allosteric modulation of the given enzyme is independent of its association-dissociation proclivities (Horton et al., 1974). The Pseudomonaceae are aerobic organisms characterized by a very active glucose oxidation (Doelle, 1969). For the species Pseudomonas aeruginosa, the Embden-Meyerhoff pathway is not functional, since the production of pyruvate takes place by using the pentose pathway (Warburg-Dickens-Horecker pathway) and the Entner-Doudoroff pathway (Stern et al., 1960). The oxidation of pyruvate could occur by means of the citric acid cycle, but it seems more likely that the glyoxylate cycle is preferred. DPNH is used in a respiratory chain which has 0’ as a terminal electron acceptor (see Figure 3) or N03- in the case of a low oxygen level (Fewson and Nicholas, 1961). It is our view that activation of PATH by 2’-AMP and related nucleotides is an in vitro property, and hence has no physiological importance (Widmer and Kaplan, 1976). Therefore, PATH can be considered as a true unidirectional catalyst. In such a case, its physiological role is to promote TPNH oxidation with a concomitant generation of DPNH. This irreversible catalysis certainly corresponds to a specific metabolic necessity. The pentose phosphate pathway and the Entner-Doudoroff pathway make use of the enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44), which are TPNf specific. In order for their reactions to proceed at an appreciable rate, TPNH has to be by some means reoxidized. That can undoubtedly occur through biosynthetic activity, but the presence of path definitely provides a more direct way of TPN+ evolution. An equally important advantage is that hydrogen can be readily transferred from T P N H to DPN+; DPNH can then enter the respiratory chain and promote ATP synthesis. Therefore, PATH might be endowed with a central role in the metabolism of the microorganism (see Figure 3). ( 1 ) It provides the oxidation power needed for glucose catabolism. (2) The direct link to the respiratory chain allows this catabolism to be a source of ATP.’ An in vivo activation by 2’-AMP and related nucleotides would suppress the unidirectional character of PATH. This would be unfavorable to the microorganism, because such an event would oppose TPN+ evolution and disturb the link between carbohydrate oxidation and respiratory chain. The regulatory enzymes, which have provided the basis for working out of the MWC model, have two common characteristics: they operate in metabolic pathways immediately after a branching point, and for each of them the specific inhibitor (allosteric effector) is the terminal metabolite of the corresponding pathway (Monod et al., 1963). This observation explains the limitative point of view according to which allosteric effectors do not have any direct chemical or metabolic relation with the possible stubstrates, coenzymes or products of the
15, NO. 21, 1976
When the enzyme was discovered, the Pseudomonas strain was grown on citrate as the sole source of carbon. I n such a case, the metabolic role of the transhydrogenase might be quite similar, because the isocitrate dehydrogenase present in the studied microorganism is a TPN+-specific enzyme (Colowick et al.. 1952).
INTERACTION OF COENZYMES WITH ISOCITRATE DEHYDROGENASE
enzyme they act upon. For PATH, the situation is exactly the opposite (the inhibitor TPN+ is a potential substrate) and can be explained by the particular metabolic role of the enzyme (see Figure 3). Unlike the above-mentioned regulatory enzymes, PATH is not the first catalyst of a multistage metabolic pathway, but the essential link between two pathways. Since the enzyme has to comply with the physiological necessity of unidirectional catalysis, the direct control by the product TPN+ appears to be the best way of regulation (if not the only possible one). The fact that TPN+ behaves as negative effector explains the irreversibility of TPN+ evolution, but is also responsible for the TPN+ inhibition of the TPNH-DPN+ reaction. The latter effect could correspond to a kind of “metabolic buffer effect” averting too large a consumption of TPNH, which is also needed for biosynthetic purposes. References Cohen, P. T. (1967), Ph.D. Thesis, Brandeis University, Waltham, Mass., Ann Arbor, Mich., University Microfilms Inc., No. 67- 16542. Cohen, P. T., and Kaplan, N. 0. (1970a), J. Biol. Chem. 245, 2825-2836. Cohen, P. T., and Kaplan, N. 0. (1 970b), J. Biol. Chem. 245, 4666-4672. Cohen, R . (1963), C. R . Hebd. Seances Acad. Sci., Ser. C 256,
3513-3515. Colowick, S. P., Kaplan, N. O., Neufeld, E. F., and Ciotti, M. M. (1952), J. Biol. Chem. 195. 95-105. Doelle, H. W. (1969), In Bacterial Metabolism, New York, N.Y., Academic Press, 352-401. Fewson, A., and Nicholas, D. J. D. (1961), Biochim. Biophys. Acta 49, 335-349. Hanson, K. R . (1966), J . Mol. Biol. 22, 405-409. Haschemeyer, R. H., and de Harven, E. (1974), Annu. Rev. Biochem. 43, 279-301. Horton, H. R., Swaisgood, H . E., and Mosbach, K . (1974), Biochem. Biophys. Res. Commun. 61, 1 1 18- 1 124. Kemper, D., and Everse, J. (1973), Methods Enzymol. 27, 67-82. Louie, D. D., Kaplan, N. O., and Mc Lean, J. D. (1972), J. Mol. Biol. 70, 65 1-664. M o d , J., Changeux, J. P., and Jacob, F. (1 963), J . Mol. Biol. 6 , 306-329. Monod, J . , Wyman, J., and Changeux, J. P. (1965), J. Mol. Biol. 12, 88-1 18. Ochoa, S. (1955), Methods Enzymol. 1 , 699-704. Stern, I. J., Wang, C. H., and Gilmour, C. M. (1960), J. Bacteriol. 79, 601 -6 1 1. Widmer, F., and Kaplan, N. 0. (1976), Biochemistry 15, (preceding paper in this issue).
Coenzyme Binding by Triphosphopyridine Nucleotide Dependent Isocitrate Dehydrogenase from Beef Liver. Equilibrium and Kinetics Studies? Marie France Carlier* and Dominique Pantaloni
ABSTRACT: The binding of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide phosphate (NADP) dependent isocitrate dehydrogenase from beef liver cytoplasm was studied by several equilibrium techniques (ultracentrifugation, molecular sieving, ultrafiltration, fluorescence). Two binding sites (per dimeric enzyme molecule) were found with slightly different dissociation constants (0.5 and 0.12 pM) and fluorescence yields (7.7 and 6.3). A ternary complex was formed between enzyme, isocitrate, and NADPH, in which NADPH dissociation constant was 5 pM, On the contrary, no binding of NADPH to the enzyme took place in the presence of magnesium isocitrate. Dialysis experiments showed the existence of 1 NADP binding site/dimer, with a dissociation constant of 26 pM. When NADPH was present with the enzyme in the proportion of 1 molecule/dimer, the dissociation constant of NADP was de-
creased fourfold, reaching a value quantitatively comparable to the Michaelis constant. The kinetics of coenzyme binding was followed using the stopped-flow technique with fluorescence detection. NADPH binding to the enzyme occurred through one fast reaction (kl = 20 pM-I s-l). Dissociation of NADPH took place upon NADP binding; however, equilibrium as well as kinetic data were incompatible with a simple competition scheme. Dissociation of NADPH from the enzyme upon magnesium isocitrate binding was preceded by the formation of a transitory ternary complex in which the fluorescence of NADPH was only about 30% of that in the enzymeNADPH complex. The interaction between the coenzymes and the involvement of ternary complexes in the catalytic rnechanism are discussed in relation with what is known about the regulatory role of the coenzyme (Carlier, M. F., and Pantaloni, D. (1976), Biochemistry 15, 1761-1766).
Previous studies (Carlier and Pantaloni, 1973) have shown that isocitrate dehydrogenase (threo-D,-isocitrate:NADP+
oxidoreductase (decarboxylation) EC 1.I . 1.42) purified from beef liver cytoplasm is a dimeric enzyme of molecular weight 48 000 X 2. In the absence of divalent metal cations, steadystate kinetics exhibit catalytic activation by NADPH, the reaction product (Carlier and Pantaloni, 1976a). It has been demonstrated that NADPH did not play a redox role in this activation and was probably involved in the second step (de-
t From the Laboratoire d’Enzymologie du C.N.R.S., 91 190 Gif-surYvette, France. Receiued May 17, 1976. Abbreviations used are: NAD, nicotinamide adenine dinucleotide; NADP, N A D phosphate; NADPH, reduced NADP; EDTA, (ethylenedinitri1o)tetraacetic acid.
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