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    J. Biochem., 77, 1047-1056 (1975)

    A Kinetic Study of the -Keto Acid Dehydrogenase Complexesfrom Pig Heart Mitochondria1

    Minoru HAMADA,* Kichiko KOIKE,* Yutaka NAKAULA,*Tadayasu HIRAOKA,* Masahiko KOIKE,* andTakashi HASHIMOTO***Department of Pathological Biochemistry , Atomic Disease Institute,Nagasaki University School of Medicine, 12-4, Sakamoto-machi,Nagasaki, Nagasaki 852, and **Department of Biochemistry,Shinshu University School of Medicine, 3-1-1 Asahi,Matsumoto, Nagano 390

    Received for publication, October 16, 1974

    The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25. A three-siteping-pong mechanism for the actin of both complexes was proposed on the basis ofthe parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvateconcentration for various levels of CoA and a level of NAD+ near its Michaelisconstant value. Rate equations were derived from the proposed mechanism.

    Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are : 2-oxoglutarate, 0.220 mM ; CoA, 0.025 mM ; NAD+, 0.050 mm.Those of the pyruvate dehydrogenase complex are : pyruvate, 0.015 mM ; CoA, 0.021mm; NAD+, 0.079 mm.Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ inboth overall reactions, and that succinyl-CoA or acetyl-CoA and NADH wereuncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However,noncompetitive (rather than uncompetitive) inhibition patterns were observed forsuccinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These resultsare consistent with the proposed mechanisms.

    The mammalian 2-oxoglutarate dehydrogenase complex (OGDC) contains a core consisting of1 molecule of lipoate succinyltransferase (E2,Site 2) to which 6 molecules each of 2-oxoglutarate dehydrogenase (E1, Site 1) and aflavoprotein, lipoamide dehydrogenase (E3, Site3) are joined (1-4 ). The complex has beenreported to catalyze the following coordinatedsequence of reactions (Eqs. 1-5)

    1 This study was supported in part by grants fromthe Ministry of Education of Japan, and the VitaminB Research Committee.Abbreviations : TPP, thiamine-PP; LipS2, lipoicacid ; succinyl-S-Lip-SH, S-succinyldihydrolipoic acid;Lip(SH)z , dihydrolipoic acid.

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    1048 M. HAMADA, K. KOIKE, Y. NAKAULA, T. HIRAOKA, M. KOIKE, and T. HASHIMOTO2-Oxoglutarate+[TPP]-E1 [Succinic semialdehyde-TPP]-E1+C02 (1)[Succinic semialdehyde-TPP]-E1+[LipS2]-E2[Succinyl-S-Lip-SH]-E2+[TPP]-E1 (2)[Succinyl-S-Lip-SH]-E2+CoA-SHSuccinyl-S-CoA+[Lip(SH)2]-E2 (3)[Lip(SH)2]-E2+[FAD]-E3[Reduced FAD]-E3+[LipS2]-E2 (4)[Reduced FAD]-E3+NAD+[FAD]-E3+NADH+H+ (5)Sum : 2-Oxoglutarate+CoA-SH+NAD+Succinyl-S-CoA+CO2+NADH+H+ (6)

    These transformations involve an interaction of the lipoyl moiety (LipS2) of lipoate succinyltransferase with the TPP bound to 2-oxoglutarate dehydrogenase and with a flavin adenine dinucleotide (FAD) bound to lipoamidedehydrogenase (2) . The pyruvate dehydrogenase complex (PDC) catalyzes the oxidativedecarboxylation of pytuvate in the same way.This complex contains 1 molecule of lipoateacetyltransferase (LAT) [EC 2.3.1.12], 30 molecules of pyruvate dehydrogenase (PDH) [EC1.2.4.1], and 6 molecules of lipoamide dehydroRenase [EC 1.6.4.31 (5).A new approach to the derivation of therate equation for the enzyme complex has recently been proposed in terms of "fractionaloccupancy" by assuming that the interactionbetween sites is kinetically important (6).

    It was first suggested by Tsai et al. (7)that the overall reaction (Eq. 6) of bovinekidney PDC proceeded via a three-site ping-pong mechanism. Reactions at the three sitesin the pig heart OGDC are not random-sequential, and good evidence is available thatthe reactions at three sites are ping-pong type,and might be expected to have the same re-action mechanism as the bovine kidney PDCreported by Tsai et al. (7).

    We can diagram such a mechanism for thepig heart OGDC as Diagram 1.This paper reports on initial velocity andproduct inhibition studies of OGDC and PDCfrom pig heart mitochondria.

    MATERIALS AND METHODSMaterials-Substrates purchased from commercial sources were used directly or aftertreatment as described previously (2-5).Acetyl-CoA (P-L Biochemicals), CoA (Boehringer Mannheim), NAD+ and NADH (Sigma)were purchased from the indicated sources.Dihydrolipoamide was prepared by reducinglipoamide with sodium borohydride (8). Succinyl-CoA was prepared by succinylation ofCoA from succinate (9, 10). Highly purified

    preparations of OGDC and PDC were obtainedfrom pig heart mitochondria as described previously (2, 5).Methods-Assays for the overall forwardreactions were carried out as described previously (2, 3, 5). The initial rate of the reaction with the complexed lipoamide dehydrogenase (E1-E2-E3) was determined by measuring the oxidation of NADH by lipoamide (Eq.7) (2, 5).Lipoamide+NADH+H?? Dihydrolipoamide+NAD+ (7)

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    KINETIC STUDY OF -KETO ACID DEHYDROGENASE COMPLEXES 1049Kinetic Studies-The assays with OGDCand PDC were run in 0.075 and 0.05 M potassium phosphate buffer (pH 7.5), respectively,

    at 25 in a Beckman DB recording spectrophotometer. Kinetic constants are defined us-ing Cleland's nomenclature (11-14 ).Analysis of Kinetic Data-The initial velocity data were plotted graphically (15) indouble-reciprocal form to check on the linearityof the relationships and secondary plots weremade of the slopes or intercepts (or both) fromthe primary plots as a function of the reciprocal of either substrate or inhibitor concentration. The data obtained from initial velocity experiments fit the general equation (Eq.8) derived by Cleland (14) for a "three-siteping-pong" mechanism in the absence of products ; K,, K,, and K are the Michaelis constants for 2-oxoglutarate or pyruvate (A), CoA(B), and NAD+ (C), respectively.

    Michaelis constants for all of the reactions, aswell as the maximum velocities, are expressed

    as averages based on several experiments.

    RESULTSInitial Velocity Studies-Initial overall veloc

    ity patterns obtained when the concentrationof one of the three substrates, 2-oxoglutarateor pyruvate, CoA, and NAD+, was varied inthe presence of various concentrations of thesecond substrate and with the concentrationof the third substrate held constant are shownin Figs. 1 and 2.Plots of 1/v against [2-oxoglutarate]-' or[pyruvate]`' at various levels of CoA and withconcentrations of NAD+ near its Michaelisconstant (0.025 or 0.050 mm, respectively) gavea series of parallel lines (Figs. 1A and 2A).Using the rate equations for the three-site ping-pong mechanism derived by Cleland (Eq. 8),the constants K, Kb, and K for OGDC wereestimated to be 0.220, 0.025, and 0.050 mm,respectively, from primary plots of reciprocalinitial velocities versus reciprocal concentrations of 2-oxoglutarate, secondary plots of primary intercepts versus reciprocal concentrations of CoA, and tertiary plots of secondary

    Fig. 1. Initial velocity patterns for the overall reaction (Eq. 6) in the 2-oxoglutaratedehydrogenase complex. A : 2-Oxoglutarate concentration was varied in the presenceof various CoA concentrations as indicated. NAD+ was held constant at 0.025 MM.B : CoA concentration was varied in the presence of various NAD+ concentrations asindicated. 2-Oxoglutarate was held constant at 2.5 mm. C : NAD+ concentrationwas varied in the presence of various 2-oxoglutarate concentrations as indicated.CoA was held constant at 0.02 mm. Assay conditions were as described under" METHODS ." The reaction medium contained, in a volume of 2 ml, 0.5 mm CaCl2,and 2.5 MM L-cysteine. The reaction was initiated by the addition of 18 jig of theOGDC preparation. Velocity is expressed as micromoles of NADH formed per min.

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    1050 M. HAMADA, K. KOIKE, Y. NAKAULA, T. HIRAOKA, M. KOIKE, and T. HASHIMOTO

    Fig. 2. Initial velocity patterns for the overall reaction (Eq. 6) of the pyruvatedehydrogenase complex. A : Pyruvate concentration was varied in the presence ofvarious CoA concentrations as indicated. NAD+ was held constant at 0.05 mm.B: CoA concentration was varied in the presence of various NAD+ concentrations asindicated. Pyruvate was held constant at 0.25 mm. C: NAD` concentration wasvaried in the presence of various pyruvate concentrations as indicated. CoA washeld constant at 0.05 mm. The reaction medium contained 1 mm MgC12, 0.2 mmthiamine pyrophosphate, and 2.6 mm cysteine HCl. The reaction was initiated bythe addition of 9 g of PDC. Other conditions were as in Fig. 1.

    intercepts versus reciprocal concentrations ofNAD+. With PDC (Fig. 2), these constantswere 0.015, 0.021, and 0.079 mm, for pyruvate,CoA, and NAD+, respectively.Product Inhibition Experiments-The rateequations derived for the three-site ping-pongmechanism (Eq. 8) predict that each productwill be competitive with respect to the substrate that binds at that site, and uncompetitive with respect to other substrates. In agreement with these predictions, succinyl-CoA andacetyl-CoA were competitive with respect toCoA (Fig. 3) and NADH was competitive withrespect to NAD+ (Fig. 4). Succinyl-CoA oracetyl-CoA and NADH were all uncompetitivewpith respect to 2-oxoglutarate or pyruvate(Figs. 5 and 6). However, noncompetitive(rather than uncompetitive) inhibition kineticswere observed for succinyi-CoA or acetyl-CoAversus NAD+ (Fig. 7) and for NADH versusCoA (Fig. 8). A product will be noncompetitive with respect to the substrate that bindsat the next site in the overall reaction sequence if the reaction at this site is random-sequential. Also, if a product is a dead-endinhibitor of a subsequent site or in some wayhinders the reaction at that site, it will be non-

    competitive with respect to the substrate thatnormally binds at this site. The anomalousinhibition patterns observed with succinyl-CoAor acetyl-CoA versus NAD+ and with NADHversus CoA could be caused by protein-proteininteraction between the lipoate succinyltransferase or lipoate acetyltransferase and the lipoamide dehydrogenase.Effects of 2-Oxoglutarate (or Pyruvate) andCoA on the Lipoamide Dehydrogenase, Flavoprotein-catalyzed Reaction (Eq. 7)-The organizations of both multienzyme complexes (El-E2-E3) suggest the possibility that protein-proteininteractions between El and E2 or E2 and E3might have an effect on the reactions catalyzedby the individual enzymes . To investigate thispossibility, a kinetic study of Eq. 7 was under-taken using the flavoprotein in association withthe lipoate succinyltransferase or lipoate acetyltransferase as an integral part of the OGDCor PDC, respectively .That no interaction occurs between El andE3 in OGDC or PDC is indicated by the observation that 2-oxoglutarate or pyruvate hadno effect on the rate of Eq . 7 (Fig. 9). How-ever, CoA inhibited the complexed flavoproteinof OGDC noncompetitively with respect to

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    KINETIC STUDY OF -KETO ACID DEHYDROGENASE COMPLEXES 1053

    Fig. 9. Effect of 2-oxoglutarate on the flavoprotein.catalyzed reaction (Eq. 7). The oxidation of NADHby lipoamide (1 mm) was measured as described under

    METHODS," in a volume of 2 ml: A, with 2 g ofthe 2-oxoglutarate dehydrogenase complex, in the.absence () and presence of 0.05 () and 0.1 mm() 2-oxoglutarate, 0.05 mm thiamine pyrophosphate,and 0.2 mm MgC12 ; B, with 1.6 g of the pyruvatedehydrogenase complex, in the absence () andpresence of 0.1 () and 0.2mM () pyruvate, 0.2mm thiamine pyrophosphate and 0.2 mm MgC12.

    NADH when the concentration of the fixedsubstrate was near its Michaelis constant (Fig.10A) ; with PDC, such an effect was not ob-served (Fig. 10C). CoA did not inhibit thecomplexed fiavoprotein with respect to lipoamide in either complex (Fig. 10B and 10D).These observations with OGDC suggest thatthe combination of CoA (or succinyl-CoA) with

    E in some way hinders the combination ofNADH (or NAD+) with E3.DISCUSSION

    The results presented in this paper indicatethat when 2-oxoglutarate or pyruvate, CoA, orNAD+ is used as a variable substrate in thepresence of various concentrations of the othersubstrates, the initial overall velocities of theenzymes (OGDC or PDC) show parallel typesof kinetics, if the intramitochondrial levels of

    Fig. 10. Effect of CoA on the flavoprotein-catalyzedreaction. A (OGDC), C (PDC) ; lipoamide concentration was varied. NADH concentration was heldconstant at 0.1 mm. The concentrations of addedCoA were 0 (0), 0.2 (v), and 0.4 mm (O). Otherconditions were as in Fig. 9. B (OGDC), D (PDC) ;NADH concentration was varied. Lipoamide concentration was held constant at 1 mm.NAD+ are used.At Sites 2 and 3, such parallel lines arecharacteristic of a ping-pong or binary complexmechanism (7), where the release of the product is initiated by the binding of the nextsubstrate.

    Let us consider the mechanism of the over-all reactions catalyzed by OGDC. The reaction at Site 1 can be written as:

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    1054 M. HAMADA, K. KOIKE, Y. NAKAULA, T. HIRAOKA, M. KOIKE, and T. HASHIMOTOwhere T and H are the two stable enzymeforms at Site 1 (TPP and succinic-semialdehyde TPP - binding 2 - oxoglutarate dehydrogenase). A and P are the substrate (2-oxoglutarate) and product (C02) concentrations re-acting at this site. However, as the reactionat this site is irreversible, the first product P-term could be dropped. S and X correspondto the oxidized and succinylated lipoate in E2,respectively.

    We can derive the rate equation as follows :

    However, at higher concentrations ofNAD 1, the kinetics for CoA versus 2-oxoglutarate with OGDC gave lines intersecting atthe abscissa (data not shown). At Site 1, both2-oxoglutarate and CoA are cooperatively essential for the release of the first product, C02.This may be interpreted as indicating that thereaction at Site 1 with bound TPP is consistentwith a random mechanism, where the combination of the second substrate at Site 2 occursbefore the first product is released from Site1.

    The reaction processes at Site 2 may beschematically represented as shown below :

    where a bound carrier has three possible formslabelled S, X, and L (corresponding to the

    oxidized, [LipS2]-E2 succinylated, [succinyl-S-Lip-SH]-E2 ; and reduced, [Lip(SH)2]-E2 formsof OGDC) ; Ox and Red correspond to exidizedand reduced FAD bound to E3, respectively.The terms f5 and f6 are the fractional occupancy factors for the other sites, given bythe following equations :

    where B is the substrate (CoA) concentrationat Site 2 ; Q is the product (succinyl-CoA) atthis site ; and the K; values are dissociationconstants.

    There is good evidence that the reactionat Site 3 in a multisite ping-pong mechanismis itself ping-pong type. In this case, the following equation must also be added :

    where Ox and Red correspond to oxidized andreduced FAD bound to E3; C and R are thesubstrate (NAD+) and product (NADH) concentrations at Site 3.We can derive equations for Ox and Red

    analogous to Equation 10.Since Red+Ox=1, if the concentrations ofOx and Red are considered as fractions of theirpotential values, then

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    KINETIC STUDY OF -KETO ACID DEHYDROGENASE COMPLEXES 1055If a three-site ping-pong mechanism iscorrect, we can derive the rate equation fromthe mechanism (12) by the method of Kingand Altman (16, 17). We obtain :

    when T, H, Ox, and Red are substituted.This transforms into:

    In this shorthand notation, 1=k1, 2=k2, etc.for the rate constants, each B is really B/Kiband each Q is Q/Kio.From this Eq. 16 and the mechanism (12),one would theoretically expect all parallel linesin double-reciprocal plots of B versus A, Cversus B, and A versus C, respectively. Succinyl-CoA or acetyl-CoA should be competitivewith respect to CoA and uncompetitive forothers, and NADH should be competitive withrespect to NAD+ and uncompetitive for others.With both complexes parallel lines were obtained in all cases, and thus the data conform-ed to the above interpretation.The. product inhibition patterns call foreach product to be competitive with the substrate combining at that site, regardless ofwhether the reaction at that site is random-sequential or ping-pong (unless central coresare kinetically important, in which case the

    ping-pong site will give a noncompetitive pat-tern and the random site a competitive one)and this has been observed.Using the uncomplexed lipoamide dehydrogenase from rat liver mitochondria, Reed (18)reported that their result was consistent withthe Bi Bi Ping-Pong mechanism originally pro-

    posed by Massey et al. (1.9) ; and in addition,the formation of kinetically significant, abortive complexes between the enzyme and NADHor lipoamide was observed.Succinyl-CoA or acetyl-CoA and NADH

    are both uncompetitive with respect to 2-oxoglutarate or pyruvate, but succinyl- or acetyl-CoA is noncompetitive with respect to NAD+,and NADH noncompetitive with respect toCoA, suggesting that each product may be adead-end inhibitor of the subsequent site, ormay in some way hinder reaction at that site.However, as the lipoamdie is actually the freeform of the lipoyl moiety, these observationsmay not accurately represent the physiologicalsituation.These investigations indicate the existenceof differences in the reaction mechanisms between OGDC-E1 and PDC-E1 at greater-than-physiological concentrations of NAD+, with orwithout bound TPP at each site. These differences may be due to the binding of TPP caus-ing the OGDC-E1 to undergo a conformationalchange or to the swing of the lipoyl moietyfrom Site 1 to Site 2. We intend to investigatethis point in more detail.We wish to thank Dr. Ken H. Tachiki, Section ofNeurobiology, Institute of Psychiatric Research,Indiana University Medical School, for his criticismsand kind help in the preparation of the manuscript.We also thank Dr. W.W. Cleland, Department ofBiochemistry, University of Wisconsin, for his criticisms.

    REFERENCES1. Hirashima, M., Hayakawa, T., & Koike, M.

    (1967) J. Biol. Chem. 242, 902-9072, Tanaka, N,, Koike, K., Hamada, M., Otsuka,K.-I., Suematsu, T., & Koike, M. (1972) J. Biol.Cheap. 247, 4043-40493. Tanaka, N., Koike, K., Otsuka, K.-I., Hamada,M., Ogasahara, K., & Koike, M. (1974) J. Biol.Chem. 249, 191-1984. Koike, K., Hamada, M., Tanaka, N., Otsuka,K.-I., Ogasahara, K., & Koike, M. (1974) J. Biol.Chem. 249, 3836-38425. Hayakawa, T., Kanzaki, T., Kitamura, T.,Fukuyoshi, Y., Sakurai, Y., Koike, K., Suematsu,T., & Koike, M. (1969) J. Biol. Chem. 244, 3660-3670

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    1056 M. HAMADA, K. KOIKE, Y. NAKAULA, T. HIRAOKA, M. KOIKE, and T. HASHIMOTO,6. Cleland, W.W. (1973) J. Biol. Chem. 248, 8353-

    83557. Tsai, C.S., Burgett, M.W., & Reed, L.J. (1973)I. Biol. Chem. 248, 8348-83528. Reed, L.J., Koike, M., Levitch, M.E., Leach,F.R. (1958) J. Biol. Chem. 232, 143-1589. Ochoa, S. (1954) in Methods in Enzymology(Colowick, S.P. & Kaplan, N.O., eds.) Vol. 1,p. 688, Academic Press, New York10. Khorana, H.G. (1959) J. Am. Chem. Soc. 81,1265

    11. Cleland, W.W. (1963) Biochim. Biophys. Acta 67,104-13712. Cleland, W.W. (1963) Biochim. Biophys. Acta 67,173-187

    13. Cleland, W.W. (1963) Biochim. Biophys. Acta 67,188-19614. Cleland, W.W. (1967) Ann. Rev. Biochem. 36,.77-11215. Dixon, M. & Webb, E.C. (1967) in Enzymes,6th impr. esp. chaps. 2, 4, 8, and 9, AcademicPress, New York16. King, E.L. & Altman, C. (1956) J. Phys. Chem..60, 1375-137817. King, E.L. (1956) J. Phys. Chem. 60, 1378-138118. Reed, J.K. (1973) J. Biol. Chem. 248, 4834-483919. Massey, V., Gibson, Q.H., & Veeger, C. (1960)Biochem. J. 77, 341-351

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