Professional Documents
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77, 1047-1056
(1975)
A from
Kinetic Pig
Study Heart
of
the -Keto
Acid
Dehydrogenase
Complexes
Mitochondria1
Kichiko
KOIKE,*
Yutaka and
NAKAULA,*
Masahiko
Biochemistry of of Medicine,
KOIKE,*
**Department Medicine,
Received
for
publication,
October
16,
1974
The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease com plexes from pig heart mitochondria were studied at pH 7.5 and 25. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism.
Michaelis plex reaction Those of the mm; NAD+, constants for the reactants of the 2-oxoglutarate dehydrogenase com are : 2-oxoglutarate, pyruvate dehydrogenase 0.079 mm. 0.220 mM ; CoA, 0.025 mM ; NAD+, 0.050 mm. complex are : pyruvate, 0.015 mM ; CoA, 0.021
Product inhibition studies showed that succinyl-CoA or acetyl-CoA was com petitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.
The
mammalian
2-oxoglutarate
dehydrogenase
1 This
study
was
supported
in part of Japan,
by grants
from
complex (OGDC) contains a core consisting of 1 molecule of lipoate succinyltransferase (E2, Site 2) to which 6 molecules each of 2-oxo glutarate dehydrogenase (E1, Site 1) and a flavoprotein, lipoamide dehydrogenase (E3, Site 3) are joined (1-4 ). The complex has been reported to catalyze the following coordinated sequence of reactions (Eqs. 1-5)
the Ministry of Education B Research Committee. Abbreviations : TPP, acid ; succinyl-S-Lip-SH, Lip(SH)z , dihydrolipoic
1047
1048
M. HAMADA,
2-Oxoglutarate+[TPP]-E1 [Succinic
K. KOIKE,
Y. NAKAULA,
T. HIRAOKA,
M. KOIKE,
and T. HASHIMOTO
(1) (2) (3) (4) (5) (6)
[Succinic
semialdehyde-TPP]-E1+C02
semialdehyde-TPP]-E1+[LipS2]-E2[Succinyl-S-Lip-SH]-E2+[TPP]-E1
These transformations involve an interac tion of the lipoyl moiety (LipS2) of lipoate suc cinyltransferase with the TPP bound to 2-oxo glutarate dehydrogenase and with a flavin ade nine dinucleotide (FAD) bound to lipoamide dehydrogenase (2) . The pyruvate dehydro genase complex (PDC) catalyzes the oxidative decarboxylation of pytuvate in the same way. This complex contains 1 molecule of lipoate acetyltransferase (LAT) [EC 2.3.1.12], 30 mole cules of pyruvate dehydrogenase (PDH) [EC 1.2.4.1], and 6 molecules of lipoamide dehydro Renase [EC 1.6.4.31 (5).
A new approach to the derivation of the rate equation for the enzyme complex has re cently been proposed in terms of "fractional occupancy" by assuming that the interaction between sites is kinetically important (6).
It was first suggested by Tsai et al. (7) that the overall reaction (Eq. 6) of bovine kidney PDC proceeded via a three-site pingpong mechanism. Reactions at the three sites in the pig heart OGDC are not random-se quential, and good evidence is available that the reactions at three sites are ping-pong type, and might be expected to have the same reaction mechanism as the bovine kidney PDC reported by Tsai et al. (7).
We pig heart can diagram OGDC as such a mechanism 1. for the Diagram
This paper reports on initial velocity and product inhibition studies of OGDC and PDC from pig heart mitochondria.
MATERIALS
AND
METHODS
Materials-Substrates purchased from com mercial sources were used directly or after treatment as described previously (2-5). Acetyl-CoA (P-L Biochemicals), CoA (Boeh ringer Mannheim), NAD+ and NADH (Sigma) were purchased from the indicated sources. Dihydrolipoamide was prepared by reducing lipoamide with sodium borohydride (8). Suc cinyl-CoA was prepared by succinylation of CoA from succinate (9, 10). Highly purified preparations of OGDC and PDC were obtained from pig heart mitochondria as described pre viously (2, 5). Methods-Assays for the overall forward reactions were carried out as described previ ously (2, 3, 5). The initial rate of the reac tion with the complexed lipoamide dehydro genase (E1-E2-E3) was determined by measur ing the oxidation of NADH by lipoamide (Eq. 7) (2, 5).
Lipoamide+NADH+H ?? Dihydrolipoamide+NAD+
(7)
J. Biochem.
KINETIC
STUDY
OF -KETO
ACID
DEHYDROGENASE
COMPLEXES
1049
Kinetic Studies-The assays with OGDC and PDC were run in 0.075 and 0.05 M potas sium phosphate buffer (pH 7.5), respectively, at 25 in a Beckman DB recording spectro
photometer. Kinetic constants are defined using Cleland's nomenclature (11-14 ). Analysis of Kinetic Data-The initial veloc ity data were plotted graphically (15) in double-reciprocal form to check on the linearity of the relationships and secondary plots were made of the slopes or intercepts (or both) from the primary plots as a function of the recip rocal of either substrate or inhibitor concen tration. The data obtained from initial veloc ity experiments fit the general equation (Eq. 8) derived by Cleland (14) for a "three-site ping-pong" mechanism in the absence of prod ucts ; K,, K,, and K are the Michaelis con stants for 2-oxoglutarate or pyruvate (A), CoA (B), and NAD+ (C), respectively.
as
averages
based
on
several
experiments.
RESULTS Initial ity or the patterns of the pyruvate, presence substrate third 1 and 2. of one Velocity obtained three CoA, and and Studies-Initial when substrates, NAD+, with held the constant the overall veloc
of various substrate
concentrations
concentration shown
Plots
of
1/v against
[2-oxoglutarate]-'
or
constants maximum
for
all
of the
as
velocities,
[pyruvate]`' at various levels of CoA and with concentrations of NAD+ near its Michaelis constant (0.025 or 0.050 mm, respectively) gave a series of parallel lines (Figs. 1A and 2A). Using the rate equations for the three-site pingpong mechanism derived by Cleland (Eq. 8), the constants K, Kb, and K for OGDC were estimated to be 0.220, 0.025, and 0.050 mm, respectively, from primary plots of reciprocal initial velocities versus reciprocal concentra tions of 2-oxoglutarate, secondary plots of pri mary intercepts versus reciprocal concentra tions of CoA, and tertiary plots of secondary
Fig. 1. Initial velocity patterns for the overall reaction dehydrogenase complex. A : 2-Oxoglutarate concentration
of 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 as indicated. 2-Oxoglutarate was held constant at 2.5 mm. C : NAD+ concentration was varied in the presence of various 2-oxoglutarate concentrations as indicated. CoA was held constant at 0.02 mm. Assay " METHODS ." The reaction medium contained, and 2.5 MM L-cysteine. OGDC preparation. The reaction Velocity is expressed conditions were as described under in a volume of 2 ml, 0.5 mm CaCl2, by the addition of 18 jig of the formed per min. of NADH
was initiated
as micromoles
Vol.
77,
No.
5, 1975
1050
M.
HAMADA,
K. KOIKE,
Y. NAKAULA,
T. HIRAOKA,
M. KOIKE,
and T. HASHIMOTO
Fig.
2.
Initial
velocity complex.
patterns A : Pyruvate as was was of mm. and of PDC. varied held various The 2.6 Other
for
the
overall
6) in
of the
the
presence at 0.05
concentrations
indicated. in the
constant
1 mm reaction Fig. 1.
MgC12, was
cysteine conditions
initiated
intercepts versus reciprocal concentrations of NAD+. With PDC (Fig. 2), these constants were 0.015, 0.021, and 0.079 mm, for pyruvate, CoA, and NAD+, respectively. Product Inhibition Experiments-The rate equations derived for the three-site ping-pong mechanism (Eq. 8) predict that each product will be competitive with respect to the sub strate that binds at that site, and uncompeti tive with respect to other substrates. In agree ment with these predictions, succinyl-CoA and acetyl-CoA were competitive with respect to CoA (Fig. 3) and NADH was competitive with respect to NAD+ (Fig. 4). Succinyl-CoA or acetyl-CoA and NADH were all uncompetitive wpith respect to 2-oxoglutarate or pyruvate (Figs. 5 and 6). However, noncompetitive (rather than uncompetitive) inhibition kinetics were observed for succinyi-CoA or acetyl-CoA versus NAD+ (Fig. 7) and for NADH versus CoA (Fig. 8). A product will be noncompeti tive with respect to the substrate that binds at the next site in the overall reaction se quence if the reaction at this site is randomsequential. Also, if a product is a dead-end inhibitor of a subsequent site or in some way hinders the reaction at that site, it will be non-
to site.
substrate
that
The
acetyl-CoA
interaction
lipoate
or lipoate
acetyltransferase
dehydrogenase.
Effects of 2-Oxoglutarate (or Pyruvate) and CoA on the Lipoamide Dehydrogenase, Flavo protein-catalyzed Reaction (Eq. 7)-The organi zations of both multienzyme complexes (El-E2E3) suggest the possibility that protein-protein interactions between El and E2 or E2 and E3 might have an effect on the reactions catalyzed by the individual enzymes . To investigate this possibility, a kinetic study of Eq. 7 was undertaken using the flavoprotein in association with the lipoate succinyltransferase or lipoate acetyl transferase as an integral part of the OGDC or PDC, respectively .
That no interaction occurs between El and E3 in OGDC or PDC is indicated by the ob servation that 2-oxoglutarate or pyruvate had no effect on the rate of Eq . 7 (Fig. 9). However, CoA inhibited the complexed flavoprotein of OGDC noncompetitively with respect to
J. Biochem.
PLEXES 1051 Fig. presence of A : The 0 glutarate mm, B : The 0 and at ditions 2.5 and were CoA (), 0.02 (), concentrations and constant at 2.5 and and CoA (), 0.05 (), and 0.10 concentrations of 0.2 mm of various concentrations added mm 4. Product inhibition by concen
Fig.
3.
Product
inhibition
by
succinyl-CoA
or
in
Fig.
Product in the
by of
succinyl-CoA various pyruvate, of (), added and 0.34 concen respec suc
or
acetyl-CoA
in
the
presence
of
various
of
CoA.
A : The
concentrations
added
succinyl-CoA
were
()
().
2-Oxoglutarate
and
NAD+
mm respectively. NADH were Pyruvate constant (). held tively. CoA CoA constant B : The were 0 () and at NAD+ 0.04 concentrations and 0.2mM concentrations and 2.5 rum, of adedd (). CoA were respec acetyland
tions
were
held
constant
at
2.5
and
respectively.
B : The
concentrations
acetyl-CoA
were
()
and
0.2
Pyruvate
and
NAD+
concentrations
constant
at
2.5
mm.
Other
conditions
Other
con
NAD* 0.065
were respectively.
held
constant
at
as
in
Figs.
and
2.
Fig. presence Fig. in or of concentrations trations (), and concentrations and 0.04 of of and were held tively. (). constant Pyruvate at 0.2 and 2.5 CoA and concentrations 0.065 mm, were respec added acetyl-CoA were 0 () and 0.2 mat mm, respectively. 1.5 B : The concentrations trations were 0.1 mM (). of added succinyl-CoA 2-Oxoglutarate held constant at of NAD+. concentra 0.05 concen 2.5 and 0.2 2-oxo acetyl-CoA in the presence A : The were and the of various concen 0 () CoA 0 () NAD+ 2.5 7. Product inhibition by succinyl-CoA
8.
Product of A : The various concentrations and 0.05 concentrations and centrations mat (). were 2.5 mm, of
inhibition
by concentrations of mat (). were respectively. added Pyruvate held constant NADH and at added
in
Fig.
6.
Product
inhibition
by
NADH
presence
of
various
concentrations
glutarate
or
pyruvate.
A :
The
tions
of
added
NADH
were
(),
and
0.1
mm
().
CoA
and
NAD+
trations
were
held
constant
at
0.04
mm,
respectively.
B : The
concentrations
added
NADH
were
(),
0.1
(),
mm
().
CoA
and
NAD+
concentrations
held
constant
at
0.04
and
2.5
mm,
respectively.
J. Biochem.
KINETIC
STUDY
OF -KETO
ACID
DEHYDROGENASE
COMPLEXES
1053
The of
measured
METHODS,"
0.2mM and
Fig. 10. Effect of CoA on the flavoprotein-catalyzed reaction. A (OGDC), C (PDC) ; lipoamide concen tration was varied. NADH concentration was held constant at 0.1 mm. The concentrations of added CoA were 0 (0), 0.2 (v), and 0.4 mm (O). Other conditions were as in Fig. 9. B (OGDC), D (PDC) ; NADH concentration was varied. Lipoamide con centration was held constant at 1 mm. NAD+ are used.
NADH when the concentration of the fixed substrate was near its Michaelis constant (Fig. 10A) ; with PDC, such an effect was not observed (Fig. 10C). CoA did not inhibit the complexed fiavoprotein with respect to lipo amide in either complex (Fig. 10B and 10D). These observations with OGDC suggest that the combination of CoA (or succinyl-CoA) with E 2 in some way hinders the combination of NADH (or NAD+) with E3.
DISCUSSION
At Sites 2 and 3, such parallel lines are characteristic of a ping-pong or binary complex mechanism (7), where the release of the prod uct is initiated by the binding of the next substrate.
Let us consider all tion reactions at Site 1 can the mechanism by OGDC. as: of the The overreac catalyzed
be written
The results presented in this paper that when 2-oxoglutarate or pyruvate, NAD+ is used as a variable substrate
presence of various concentrations of the other substrates, the initial overall velocities of the enzymes (OGDC or PDC) show parallel types of kinetics, if the intramitochondrial levels of
Vol.
77,
No.
5,
1975
1054
M. HAMADA,
K. KOIKE,
Y. NAKAULA,
T. HIRAOKA,
M. KOIKE,
and T. HASHIMOTO
where T and H are the two stable enzyme forms at Site 1 (TPP and succinic-semialde hyde TPP - binding 2 - oxoglutarate dehydro genase). A and P are the substrate (2-oxo glutarate) and product (C02) concentrations reacting at this site. However, as the reaction at this site is irreversible, the first product Pterm could be dropped. S and X correspond to the oxidized and succinylated lipoate in E2, respectively.
We can derive the rate equation as follows :
oxidized, [LipS2]-E2; succinylated, [succinyl-SLip-SH]-E2 ; and reduced, [Lip(SH)2]-E2 forms of OGDC) ; Ox and Red correspond to exidized and reduced FAD bound to E3, respectively. The terms f5 and f6 are the fractional oc cupancy factors for the other sites, given by the following equations :
where B is the substrate (CoA) concentration at Site 2 ; Q is the product (succinyl-CoA) at this site ; and the K; values are dissociation constants.
There at Site 3 in is itself lowing is good ping-pong equation evidence type. must also In be that this the case, : reaction the fol a multisite ping-pong added mechanism
However, at higher concentrations of NAD 1, the kinetics for CoA versus 2-oxoglu tarate with OGDC gave lines intersecting at the abscissa (data not shown). At Site 1, both 2-oxoglutarate and CoA are cooperatively es sential for the release of the first product, C02. This may be interpreted as indicating that the reaction at Site 1 with bound TPP is consistent with a random mechanism, where the combi nation of the second substrate at Site 2 occurs before the first product is released from Site 1.
The reaction processes as at Site shown 2 may below : be schematically represented
where Ox and Red correspond to oxidized and reduced FAD bound to E3; C and R are the substrate (NAD+) and product (NADH) concen trations at Site 3.
We analogous can derive equations 10. for Ox and Red to Equation
Since Red+Ox=1, if the concentrations of Ox and Red are considered as fractions of their potential values, then
where a bound carrier has three possible forms labelled S, X, and L (corresponding to the
J. Biochem.
KINETIC
STUDY
OF -KETO
ACID
DEHYDROGENASE
COMPLEXES
1055
If a three-site ping-pong mechanism is correct, we can derive the rate equation from the mechanism (12) by the method of King and Altman (16, 17). We obtain :
posed by Massey et al. (1.9) ; and in addition, the formation of kinetically significant, abor tive complexes between the enzyme and NADH or lipoamide was observed.
Succinyl-CoA are both or acetyl-CoA with respect and NADH to 2-oxo or acetylto NAD+, respect may be to a uncompetitive
dead-end inhibitor may in some way However, when This T, H, Ox, and Red are substituted. form may transforms into: not These of differences tween OGDC-E1 as the lipoyl of the
subsequent site, or reaction at that site. is actually these the free observations
accurately
physiological concentrations without bound TPP at each ences ing the Site point may or be due to the to the swing 2. We detail. OGDC-E1 1 to Site in more to undergo
of NAD+, with or site. These differ of TPP lipoyl causmoiety a conformational to investigate
In this shorthand notation, for the rate constants, each and each Q is Q/Kio.
From this Eq. 16 and the mechanism (12), one would theoretically expect all parallel lines in double-reciprocal plots of B versus A, C versus B, and A versus C, respectively. Suc cinyl-CoA or acetyl-CoA should be competitive with respect to CoA and uncompetitive for others, and NADH should be competitive with respect to NAD+ and uncompetitive for others. With both complexes parallel lines were ob tained in all cases, and thus the data conformed to the above interpretation.
The. product inhibition patterns call for each product to be competitive with the sub strate combining at that site, regardless of whether the reaction at that site is randomsequential or ping-pong (unless central cores are kinetically important, in which case the ping-pong site will give a noncompetitive pattern and the random site a competitive one) and this has been observed. Using the uncomplexed lipoamide dehydro genase from rat liver mitochondria, Reed (18) reported that their result was consistent with the Bi Bi Ping-Pong mechanism originally pro-
We wish to thank Dr. Ken H. Tachiki, Section of Neurobiology, Institute of Psychiatric Research, Indiana University Medical School, for his criticisms and kind help in the preparation of the manuscript. We also thank Dr. W.W. Cleland, Department of Biochemistry, University of Wisconsin, for his criti cisms.
REFERENCES
1. Hirashima, M.,
Hayakawa,
T.,
M.
(1967) J. Biol. Chem. 242, 902-907 2, Tanaka, N,, Koike, K., Hamada,
Otsuka,
K.-I., Suematsu, T., & Koike, M. (1972) J. Biol. Cheap. 247, 4043-4049 3. Tanaka, N., Koike, K., Otsuka, K.-I., Hamada, M., Ogasahara, K., & Koike, M. (1974) J. Biol. Chem. 249, 191-198
4. Koike, K., Hamada, M., Tanaka, N., Otsuka, K.-I., Ogasahara, K., & Koike, M. (1974) J. Biol. Chem. 249, 3836-3842
5. Hayakawa, Fukuyoshi, T., & Koike, 3670 T., Kanzaki, T., Kitamura, T., Y., Sakurai, Y., Koike, K., Suematsu, M. (1969) J. Biol. Chem. 244, 3660-
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13. Cleland, W.W. (1963) Biochim. Biophys. Acta 67, 188-196 14. Cleland, W.W. (1967) Ann. Rev. Biochem. 36,. 77-112
15. Dixon, M. & Webb, E.C. (1967) in Enzymes, 6th impr. esp. chaps. Press, New York 16. King, E.L. & Altman, 2, 4, 8, and 9, Academic C. (1956) J. Phys. Chem..
M.E.,
F.R. (1958) J. Biol. Chem. 232, 143-158 9. Ochoa, S. (1954) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) p. 688, Academic Press, New York Vol. 1,
60, 1375-1378 17. King, E.L. (1956) J. Phys. Chem. 60, 1378-1381 18. Reed, J.K. (1973) J. Biol. Chem. 248, 4834-4839 19. Massey, V., Gibson, Q.H., & Veeger, C. (1960) Biochem. J. 77, 341-351
J. Biochem.