Nucleotide Activation of Cauliflower a-Ketoglutarate Dehydrogenase by bestt571

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									        THE     JOURNAL       OF B~omorca~          CHEMISCRY
 Vol.   246,   No.   6, Issue      of March   25, pp.   1638-1643,   1971
                           Printed     in U.S.A.




Nucleotide      Activation     of Cauliflower
a-Ketoglutarate         Dehydrogenase
                                                                                                                                        (Received      for publication,       October      23, 1970)

                      RANDOLPH                T. WEDDING                AND   M.    KAY BLACK
                      From the Department                        of Biochemistry,    University      of   California,      Riverside,       California      9660.2




                                               SUMMARY                                                    frequently     observed when the enzyme being controlled                        does not
     The complex of cr-ketoglutarate                  dehydrogenase                with lipoyl            use any of the nucleotides             in its reaction.        It is believed that the
                                                                                                          control site, at which the nucleotide                  binds to produce its activa-
transsuccinylase            isolated from cauliflower                florets and assayed
with exogenous            lipoyl dehydrogenase,                 is markedly          activated            tion or inhibition,        is separate from the active site (2).
by adenosine          5’-monophosphate.                 This activation             shows an                  Enzymes associated with the tricarboxylic                    acid cycle are prom-
optimum        at about 1 mu AMP,                    with higher             concentrations               inent among those which have been shown to be controlled                                  by
                                                                                                          energy charge (3). Of particular                  interest in relation to the work




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producing       a smaller increase in rate.                   Kinetic       analysis shows
the activation          to be of the “coupling”                      type in which the                    being reported here is the demonstration                    that pyruvate dehydro-
activator       binds         to the enzyme-substrate                       complex.         The          genase from Escherichiu coli is susceptible to energy charge con-
                                                                                                          trol (2). Schwartz and Reed (4) have reported that the E. coli
presence of AMP causes an increase in the maximal                                      velocity
                                                                                                          pyruvate      dehydrogenase             is inhibited       only by guanosine             tri-
of the reaction,             measured         with variable             concentrations          of
                                                                                                          phosphate      and not by ATP, CTP, or UTP in the absence of
 cr-ketoglutarate,          and decreases the apparent                      K, for cr-keto-
                                                                                                          acetyl-CoA.        In the presence of this inhibitory                   product of the
glutarate.
     With the use of the parameters                        VL, (maximal                velocity
                                                                                                          reaction,     E. co&i pyruvate            dehydrogenase          responds to energy
                                                                                                          charge, with adenine, guanosine,                   and cytosine nucleotides.               It
at infinite      activator       concentration)         and Kz (K, for cu-keto-
                                                                                                          was found (4) that AMP, CMP, and GMP produced a 10 to 30%
glutarate       at infinite        activator      concentration)              and the ratio
                                                                                                          increase in the rate of E. coli pyruvate dehydrogenase                        activity as
 v;,:     Kz (designated             as an activation                coefficient),        it has
                                                                                                          compared with preparations                 containing      no acetyl-CoA       when the
been possible to compare                   the activation           by AMP with other
                                                                                                          nucleotides     were added in the presence of 0.1 pmole of acetyl-
nucleotides        and related compounds.                    This comparison              shows
                                                                                                          CoA.      In the experiments              of Schwartz and Reed, acetyl-CoA
that AMP          is 10 times more effective                      than ADP, which in
                                                                                                          reduced the rate in the absence of nucleotide                        to about 20% of
turn is about twice as effective as ATP, which produces                                        an
                                                                                                          that of the untreated              controls.       Shen et al. (2) reported that
activation       coefficient       about twice that of the untreated                         con-
                                                                                                          E. coli pyruvate dehydrogenase                 was stimulated        about 2-fold by 5
trol.
     This method             of comparison           of activating             effectiveness              InM    AMP,     with a somewhat greater effect when added in the
                                                                                                          presence of acetyl-CoA.
reveals      that adenosine              nucleotides          are preferred            by this
                                                                                                               The present report extends to ol-ketoglutarate                    dehydrogenase,
 enzyme, that the 2’ and 3’ -OH                      groups on the ribose of the
                                                                                                          an enzyme considered to be similar in some respects to pyruvate
nucleotide        are involved          in producing           a maximal           activation,
                                                                                                          dehydrogenase          (5), the observation            of activation      by adenosine
and that both the purine or pyrimidine                        ring and the phosphate
                                                                                                          phosphates       and provides a comparison                   of the effectiveness of
 are necessary             for maximal            effect.        Inorganic         phosphate
                                                                                                          several nucleoside monophosphates                    in activating     ar-ketoglutarate
produces       a significant        activation.
                                                                                                          dehydrogenase           from cauliflower.             This work uses an assay
     Hill plots of the rate of this reaction against cu-ketoglutarate
                                                                                                          method      in which cauliflower               ol-ketoglutarate        dehydrogenase-
 concentration         have a slope of 1, which is not altered by the
                                                                                                          lipoyl transsuccinylase            complex is coupled with saturating                 levels
presence       of nucleotide          activators.         Hill plots of rate against
                                                                                                          of pig heart lipoyl dehydrogenase                 (6) so that the reaction may be
 nucleotide       concentration          also give a slope of 1.
                                                                                                          followed by observation             of DPN reduction.             This method avoids
                                                                                                          the use of electron acceptors such as ferricyanide                        as well as the
                                                                                                          uncertain      kinetic       results obtained           when the a-ketoglutarate
                                                                                                           dehydrogenase         is purified       with a less than saturating              level of
                                                                                                           lipoyl dehydrogenase           attached to the remainder              of the complex.
   A variety of enzymes have been found to be activated         or in-                                    This makes possible a kinetic evaluation                      of such phenomena            as
hibited    by nucleoside  phosphates.     These effects have been                                         nucleotide     activation       and inhibition         of the first two enzymes in
generalized by Atkinson    (1) into a concept designated as control                                        the reaction sequence of the complex.
through     the energy charge of the cell.     In this concept, the
                                                                                                                                     EXPERIMENTAL           PROCEDURE
energy charge is low when nucleoside monophosphates         predomi-
                                                                                                          MtdTialS
nate and high when the triphosphates     are in greatest abundance.
Enzymes which are part of systems regenerating       ATP are usually                                        The cr-ketoglutarate    dehydrogenase-lipoyl      transsuccinylase
found to be activated by AMP and inhibited       by ATP, and this is                                      complex was purified from the floral heads of cauliflower (Brassica

                                                                                                     1638
Issue     of March       25, 1971                                    R. T. Wedding and M. K. Black                                                                             1639

oleraceae, L., var. botrytis)            by the methods previously                 described
 (6). The preparations               used in the work reported                 here had a
specific activity        of 2.9 to 3.1 units per mg, representing                      an ap-
proximately         50-fold purification           from the sonically              disrupted
cauliflower mitochondria.
    Commercial         pig heart lipoyl dehydrogenase                  from Boehringer
Mannheim,          dialyzed before use to remove ammonium                             sulfate,
had a specific activity             of 100 units per mg when assayed with
lipoamide       after dialysis.
    Diphosphopyridine               nucleotide,         thiamine         pyrophosphate,
lipoic acid, lipoamide,           ribose 5-phosphate,             adenosine, and AMP
were obtained            from Sigma.            Dithiothreitol,         N-tris(hydroxy-
methyl)methyl-2-aminoethane-sulfonic                       acid, ADP, GMP, CMP,
UMP,        dAMP,       and cyclic 3’,5’-AMP                 were from Calbiochem.
Coenzyme A, cy-ketoglutaric                acid, ATP, and 3’-AMP                 were from
Boehringer        Mannheim.           The biochemicals             were of the highest
                                                                                                    01                    I                     I                I                  I
purity offered.          Solutions      were adjusted to pH 7.0 with KOH,                                0                2                                      6                  8
with the exception of DPN,                 which was adjusted to pH 5.5 with                                                         [AblPj4        mM)
 KOH.        The concentration           of ADP, measured by absorbance at
                                                                                                    FIG.   1. Activation   of cauliflower a-ketoglutarate  dehydrogenase
260 nm, was assayed enzymatically                       with pyruvate           kinase and       by adenosine 5’-monophosphate.           Rates were measured with the
lactate dehydrogenase.                Contamination             with AMP was unde-               standard assay procedure described in the text with a-ketoglu-
tectable.       Solutions      of CoA were prepared fresh daily in 0.01 M                        tarate at a concentration         of 0.1 mu and additions    of AMP as




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dithiothreitol,       without pH adjustment.                                                     indicated    in the figure.    The rates are expressed as 10-s moles of
                                                                                                 DPN reduced per min in a l.O-ml assay volume.
Methods
   Assay of cw-Ketoglutarate Dehydrogenase-The                        activity       of the      and 1 pmole of dithiothreitol            in 1.0 ml final volume.           The reac-
a-ketoglutarate           dehydrogenase-lipoyl          transsuccinylase          complex        tion, which was started with lipoyl dehydrogenase,                        was linear
was determined           in an assay mixture which contained 5 pmoles of                         with enzyme          concentration.        Dialyzed        lipoyl   dehydrogenase,
N-tris(hydroxymethyl)methyl-2-aminoethane-sulfonic                             acid (pH          stored on ice, was used within 1 week after dialysis.                      Assays of
7.0), 10.5 pmoles of MgC&, 5 pmoles of DPN,                          10 pmoles of di-            cr-ketoglutarate       dehydrogenase         in the presence of 5 pmoles of
thiothreitol,       0.236 pmole of thiamine              pyrophosphate,          levels of       ar-ketoglutarate        with varying        levels of lipoyl         dehydrogenase
a-ketoglutarate         and nucleotides         as indicated     in the text, and 25             indicated that there was no further increase in rate after addition
units of pig heart lipoyl dehydrogenase.                     Each cuvette contain-               of 20 units of lipoyl dehydrogenase.
ing all reaction components               except enzymes and CoA was gently                          Lipoyl dehydrogenase            was also assayed in 1.0 ml containing
sparged with humidified                nitrogen     gas for 30 set before being                  2.5 pmoles of DPN, 1 pmole of reduced lipoic acid, 20 pmoles of
pla.ced in the cuvette chamber of a Gilford recording                              spectro-      N-tris(hydroxymethyl)methyl-2-aminoethane-sulfonic                         acid, pH
photometer          (Gilford     Instrument        Laboratories,        Inc., Oberlin,           7.0, in the presence and absence of AMP.                     These assays were run
Ohio).        The cuvette         chamber        was continually          flushed with           under Nz as described above.
humidified        Nz, and the temperature                 was maintained            at 25”.          Xtatistical    Methods-Initial         reaction      rates are expressed as
                                                                                                 M x 10-S DPNH            produced per min.            Effects of various nucleo-
After transfer to the chamber, 50 ~1 of a mixture                         of oc-ketoglu-
tarate dehydrogenase             (0.01 unit) plus lipoyl dehydrogenase                   (25     tides on the activity           of a-ketoglutarate-lipoyl         transsuccinylase
units) containing          0.5 pmole of MgCIZ and 0.011 pmole of thiamine                        were determined          with at least four ar-ketoglutarate              concentra-
pyrophosphate           were added to the cuvette.               After 30 to 45 set,             tions and three or more effector                      concentrations.       Primary
the reaction was started with 50 ~1 of a solution containing                          0.086      Lineweaver-Burk          lines were fitted according to the method of
pmole of CoA and 0.5 pmole of dithiothreitol.                        Final assay vol-            Wilkinson        (7) programmed         for a Wang model 370 computer
ume was 1.0 ml, and final pH was 7.0. Appearance                               of DPNH            (Wang Laboratories,           Inc., Tewksbury,          Massachusetts)       to pro-
was followed at 340 nm.                                                                          vide weighted fits.          Secondary plots were fitted by the method
    Since the cauliflower          a-ketoglutarate         dehydrogenase          contains       of least squares with the use of a computer program.
no thiamine          pyrophosphate          after purification       (6), addition         of
                                                                                                                                      RESULTS
MgC& and thiamine               pyrophosphate         to the enzyme mix at the
concentrations          present in the final assay assured linear initial                            The response of cauliflower cr-ketoglutarate         dehydrogenase       to
rates whether the reaction                 was started with enzyme or CoA.                       AMP in concentrations           up to 7.5 mM is illustrated        in Fig. 1.
Activation       by AMP was not affected by the order of additions;                              This experiment,      carried out at a low (0.1 mM) concentration            of
reactions were routinely              started with CoA because the higher                        a-ketoglutarate,     shows a strong activation     with a maximal       effect
viscosity of the enzyme mix made rapid addition                              and mixing          at 1.0 mM AMP, where the rate is almost 6 times as fast as that
more difhcult.                                                                                   found in the absence of AMP.             This is a peak response, and
    Lipoyl Dehydrogenase Assay-Pig                   heart lipoyl dehydrogenase                  higher concentrations        of AMP result in significantly      less activa-
(25 mg per ml) was dialyzed against three changes of 0.2 mM Tris                                 tion.    Experiments       using higher concentrations      of AMP than
buffer containing          0.05 mM EDTA, pH 7.2, to remove ammonium                              those shown have indicated a tendency for the activation              caused
sulfate.       Activity      was assayed in the presence of 5 pmoles of                          by AMP to level out at a rate approximately            three times that of
N-tris(hydroxymethyl)methyl-2-aminoethane-sulfonic                              acid (pH         the control, so that it cannot be anticipated         that the downward
7.0), 2 pmoles of lipoamide             in 95% ethanol, 0.15 pmole of DPNH,                      trend shown in this figure would result in inhibition          at very high
 1640                                                 Nucleotide       Activation        of cr-Ketoglutarate          Dehydrogenase                                          Vol.     246, No.        6

                                                                                                   of cauliflower          KG-dehydrogenasel                  by AMP is similar to that
                                                                                                   found with the E. coli pyruvate dehydrogenase.
                                                                                                       The interaction          of AMP activation               of KG-dehydrogenase               with
                                                                                                   ar-ketoglutarate          is illustrated       in Fig. 2. The large figure shows a
                                                                                                   double reciprocal plot of rate against cr-ketoglutarate                                concentra-
                                                                                                   tion over a 50-fold range.                  It is apparent from the lines for con-
         3
                                                                                                  trol and AMP concentrations                      from 0.01 to 0.3 mM that there is a
                                                                                                   progressive        stimulation         of the activity             of the enzyme by the
                                                                                                  nucleotide.          The primary plot also illustrates                     some of the charac-
                                                                                                  teristics of this stimulation.                    It may be seen that one effect of
                                                                                                  the presence of AMP is to increase V,,, of the reaction, with
                                                                                                  lines intercepting           at smaller values of l/v, and since the slopes of
                                                                                                  the lines decrease with increasing                         concentrations          of AMP,       it is
                                                                                                  apparent that the K, of KG-dehydrogenaae                               for cr-ketoglutarate          is
l/v      2                                                                                        being decreased by the AMP.
                                                                                                      This tendency for the lines of the double reciprocal                                    plot to
                                                                                                  approach a minimal                  slope at higher concentrations                     of AMP is
                                                                                                  also indicative          of the nature of the interaction                      of AMP with 01-
                                                                                                  ketoglutarate          and the enzyme.                    The theoretical           treatment       of
                                                                                                  activation        kinetics by Friedenwald                    and Maengwyn-Davies                   (8)
                                                                                                  postulates        that a l/v versus l/[S] line at infinite                         concentration




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          1                                                                                       of activator         will have a zero slope only if the activation                              is of
                                                                                                  the “coupling”            type; that is, if the activator                    promotes associa-
                                                                                                  tion of the enzyme with the substrate.                              A further requirement
                                                                                                  for the zero slope situation,                  which is approached in Fig. 2, is for
                                                                                                  the coupling          to be of the type in which the activator                             will not
                                                                                                  form a complex with the enzyme unless the substrate is already
                                                                                                  attached,       i.e. the dissociation              constant of the enzyme-activator
                                               I           I              I                       complex       is infinite.           The fact that an AMP concentration                             of
          0                 I                                                             1       0.5 mM, not shown in Fig. 2, results in a double reciprocal line
              0             2                 4            6              6              10
                                                                                                  with a slope of less than 0.01, compared with a slope of 0.27 for the
                                 l,&Ketoglutarate]             (mM)                               control line, is an indication                that the mechanism               of the activation
                                                                                                  of cr-ketoglutarate            dehydrogenase             by AMP approaches the theo-
     FIQ. 2. Interaction       of AMP with ol-ketoglutarate                in the activa-
                                                                                                  retical model in which an activator is obligatorily                               coupled to the
 tion of cauliflower         cr-ketoglutarate          dehydrogenase.           The main
jigure is a plot of the reciprocal          of rate, determined          with the stand-          enzyme-substrat,e             complex and results in an increase in the affin-
 ard assay and expressed             as in Fig. 1, against the reciprocal                    of   ity of the enzyme for its substrate.                             This latter point is sup-
or-ketoglutarate        concentration,       which was varied over the range                      ported further by the fact that AMP brings about a 13.fold
 from 0.1 to 5.0 mM. The individual                 lines represent,         respectively:        decrease in the K, for ol-ketoglutarate.
  l , control, no added AMP; n , 0.01 ~YX aP;                       A, 0.05 &         AMP;
 *, 0.1 mM AMP; +, 0.2 mM AMP; and V, 0.3 mM AMP.                                   Inset A,          The secondary plots shown as insets in Fig. 2 illustrate                               the fact
 the K, values derived             from the Wilkinson              (7) weighted          lines    that the slope of the fitted lines from the primary plot are linear
 fitted to each of the five AMP lines in the primary                  figure are plotted           against the reciprocal               of activator          concentration,           as is the K,
 against the reciprocal         of AMP concentration.                The intercept           of   for cr-ketoglutarate               determined          from the lines of the primary
 lines fitted to those points represents                   KL, the K,,, for a-keto-
                                                                                                  plot.     These secondary                 plots also demonstrate                    the methods
 glutarate     at infinite AMP concentration.                  Inset B, the slopes of
 the lines for the individual           AMP concentrat,ions              in the nrimarv           used in obtaining                the values for Vz&.JK:                     (the reciprocal         of
 figure are plotted        against the reciprocal            of AMP concentration.                the intercept          of the slope versus l/[AMP]                      line of Inset B) and
 The reciprocal       of the intercept        ofthe      line fitted to these points              KE (the intercept               of the K, versus l/[AMP]                     line of Inset A).
 provides     VC,,,: KL, a measure          of the effectiveness            of activation         Similar     secondary plots of the intercepts                        of the lines of the pri-
 by AMP.         Similar plots of the intercepts              of the lines of the pri-
 mary plot against the reciprocal              of AMP concentration               provided        mary plot against l/[AMP]                       provided         VL,,, and the ratio cal-
 VL.                                                                                               culated from the KE and VL,, provided an independent                                          check
                                                                                                   of the values obtained                   from plots of slope verms l/[AMP].
AMP concentrations.        The response to AMP shown in Fig. 1                                    The first two parameters                    correspond to the K, for cr-ketoglu-
differs somewhat from that found by Schwartz and Reed (4)                                         tarate at infinite activator                concentration           (KE) and the V,,, at
with E. coli pyruvate                                                                             infinite    activator          concentration             (Vz,,),      and the ratio V::lax:
                           dehydrogenase,        where nucleoside     mono-
phosphate relief of acetyl-CoA      inhibition    appears to be a saturat-                        KL or “activation              coefficient,”        gives a measure             of the increased
ing process without     any indication       of less activation    by higher                      velocity caused by activator                    relative to the binding of substrate
nucleotide concentrations.      Shen et al. (2) reported about a 2-fold                           and integrates the activator                   effects on both V,,,,, and K,.
                                                                                                      The Friedenwald                 and Maengwyn-Davies                     model (8), which
activation  of E. coli pyruvate dehydrogenase          by 5.0 mM AMP in
                                                                                                  appears to fit these data, was set up before allosteric control of
the absence of acetyl-CoA      with 0.05 mM pyruvate,           but they do
not present data showing the effects of other concentrations           of the                        1 The abbreviation            used is: KG-dehydrogenase,                   a-ketoglutarate
nucleotide.    In general, however, it appears that the activation                                dehydrogenase.
Issue of March       25, 1971                             R. T. Wedding and M. K. Black                                                                                  1641

                                                                                                                    TABLE     I
                                                                                         Comparison   of the effects of various nucleotides and related
                                                                                                  compounds on V,,,,, and K,,, of cauli$cwer
                                                                                                                  or-ketoglutarate         dehydrogenase
                                                                                     Values of Vl,, (maximal     velocity at infinite activator        concen-
                                                                                 tration),  K.$ (Km for a-ketoglutarate     at infinite activator concen-
                                                                                 tration)   and the ratio Vkar:KN ,,, were obtained          as indicated     in
                                                                                 Fig. 2 and in the text.      For the untreated        control,   the values
                                                                                 given are observed Vm,, and K,.             The data for experiments
                                                                                 run on different days were normalized        by the use of a standard
                                                                                 untreated    control line run at the same time.            Standard     errors
                                                                                 for V”mar and Kz were calculated       by the method of Wilkinson          (7).

                                                                                          Nucleotide                                                  K:,           V&lx: K:,
                                                                                                                       10-s   df/min                  m‘w
                                                                                 None ...........                  1.180      f    0.031      0.269   f     0.014     4.38
                                                                                 AMP ............                  3.696      f    0.087      0.021   f     0.007   197.42
                                                                                 dAMP ...........                  0.913      f    0.088      0.066   f     0.005    13.83
                                                                                 3’-AMP.          .........        1.004      f    0.049      0.418   f     0.021     2.40      ’
                                                                                 3’,5’-AMP             .......     0.920      f    0.124      0.203   f     0.036     4.53
                                                                                 ADP. ............                 1.559      f    0.163      0.096   f     0.022    18.20
                                                                                 ATP .............                 0.636      f    0.281      0.063   f     0.044    10.55
                                                                                 GMP ............                  0.894      f    0.029




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                                                                                                                                              0.083   f     0.006    10.83
                                                                                 IMP. ............                 1.559      f    0.067      0.109   f     0.018    14.25
                                                                                 UMP ............                  1.984      f    0.084      0.288   f     0.036     6.89
                                                                                 CMP ............                  1.768      f    0.012      0.111   f     0.018     7.69
           -1                                                                    Pi ...............                1.106      f    0.010      0.144   f     0.077     7.60
                -1                      0                                        Adenosine .......                 1.118      f    0.062      0.262   f     0.026     4.50
                                                                                 Ribose-5-P.             ......    1.107      f    0.049      0.265   f     0.022     4.27
                            Log [a-Ketoglutarate]                                                                                             __~

   FIG. 3. Hill plots of cauliflower     or-ketoglutarate    dehydrogenase
                                                                                 fitted to the data for the various nucleotides                         tested.       These
activity   in the presence and absence of AMP.               Concentrations
of or-ketoglutarate   are millimolar.       Assay was performed         by the   also gave slopes which did not differ significantly                         from 1 and
standard procedure described in the text.              Lines were fitted by      which did not change with alterations                      in oc-ketoglutarate         con-
the method of least squares.          0, control, no added AMP; l , 0.1          centration.        It therefore appears that, if one interprets                  the Hi11
mu AMP; 0,5 mu AMP.                                                              number as indicative             of cooperativity       between sites, there is no
                                                                                 indication      of cooperativity         between the Lu-ketoghrtarate            site and
 enzyme activity        was recognized.    One would intuitively       expect,   the putative         activator      site which binds nucleotide               phosphate.
 however, that an activator         which binds only to an enzyme-sub-           We may therefore              conclude that, at least with respect to cy-
strate complex and which increases the affinity of the enzyme for                ketoglutarate,         there is no induction            of cooperativity         between
its substrate would act through a change in the nature of the                    sites resulting       from the presence of nucleotide                 in the reaction
active site induced by the presence of the activator,              perhaps at    medium.
another site. Although           pyruvate    dehydrogenase      has been re-          In Table I are presented values of I’:%,, Kz, and the ratio
ported to exhibit sigmoid kinetics (9, lo), the cauliflower KG-de-               VZaa: K: for an array of purine and pyrimidine                              nucleotides.
hydrogenase does not show a sigmoid response to cr-ketoglutarate                 These values were obtained by extrapolation                      of secondary plots
even over a very wide concentration            range with the assay proce-       of slopes and intercepts from double reciprocal primary                           plots of
dure used in these studies.                                                      rate versus cY-ketoglutarate              concentration      at various concentra-
    This lack of a sigmoid response to ar-ketoglutarate              is shown    tions of nucleotide.           When one compares these nucleotides with
in the Hill plots of Fig. 3, in which the line without added AMP                 respect to their effectiveness in activating                    cauliflower       KG-de-
gives a slope, obtained by line fitting, of 0.99. The effect of AMP              hydrogenase,         using the integrated             effect shown in the ratio
is illustrated     by the other lines, in which the addition         of either   v;&      Kz, two facts stand out.                 One is the preference of this
0.1 mDrl AMP (slope, 1.11) or 5.0 mM AMP (slope, 1.01) does not.                 enzyme for adenosine nucleotides                   as activators,      and the other
significantly      alter the slope of a Hill plot against substrate.             the relatively           greater      effectiveness      of the monophosphate.
The two lines with AMP treatment,               which are from an experi-                                 *
                                                                                 The ratio VE,, .Kz is increased about 40-fold by AMP                                     as
ment separate from that illustrated             in Fig. 2, do demonstrate        compared with the control value.                  This is at least lo-fold greater
the effect of AMP on the reaction Km. This same absence of any                   activation     than is provided by any of the other nucleotides tested.
influence on the slopes of Hill plots was found with all levels of               It is apparent        that all three of the adenosine 5’-phosphates                     are
AMP used in these studies.            The concentration      range was from      capable of activating             the enzyme, but distinct             differences are
0.01 to 10 mM.            The other nucleotides     listed in Table I were       found in the order AMP > ADP > ATP.
equally without effect on the slopes of Hill plots against cy-keto-                   Other purine and pyrimidine                mononucleotides         and inorganic
glutarate      concentration.                                                    phosphate       also bring about some activation,                 although      substan-
    Hill plots of log v/(V,,,      - v) against log [nucleotide]     were also   tially less than AMP.               Cyclic 3’,5’-AMP,         adenosine, and ribose
1642                                                 Nucleoticle Activation              of a-Ketoglutarate          Dehydrogenase                                        Vol.    246, No.       6


5-phosphate have no significant effect on the reaction, and adeno-                                                                         DISCUSSION

sine 3’-monophosphate                significantly       inhibits      the reaction,        in-        It is difficult to compare the activation                  of cauliflower cr-keto-
creasing the Km. The other monophosphates                                   are much less          glutarate       dehydrogenase           by nucleotides          with the previously
effective than AMP and, on the basis of the indicated                              standard        reported effects of nucleotides             on E. coli pyruvate dehydrogenase
errors, are probably               significantly       less effective          than ADP.           (2, 4), partly because most of the reported data relate to over-
There appear to be differences among the nucleotides                             other than        coming the effect of an inhibitory                      product,       acetyl-CoA,         and
AMP with respect to whether the primary                            effect is on Kz or              partly because parameters                 such as K: or Vz,,:Kz                    have not
CL,      but a more extended study would be required to elucidate                                  been reported for the pyruvate dehydrogenase.                            In the few cases
any such differences.                                                                              in which direct comparison                of the activating           effect of a specific
    A few indications         of the requirements          for binding of the nucleo-              concentration         of AMP on the two enzymes is possible, it appears
tide are apparent from these data.                     The -OH            group on the 2’          that the activating            effect of an optimum           concentration         of AMP
carbon of the ribose appears to be involved                         in optimum        activa-       (-1 mu) is substantially                greater than with E. coli pyruvate
tion, since dAMP is a much poorer effector for the reaction than                                   dehydrogenase.             Even at the levels used with the E. coli pyru-
AMP.         The -OH          on the 3’ ribose carbon must also be required,                       vate dehydrogenase               (5 mM), the reaction is enhanced about 4-
since cyclic 3’,5’-AMP             is without activating             effect, and 3’-AMP            fold with the cauliflower enzyme as compared with the approxi-
actually      inhibits     the enzyme reaction.                The phosphate           group       mate 2-fold increase reported with the E. coli enzyme (2). The
appears to be a requirement                since Pi produces a small but signifi-                  inhibition     of pig heart pyruvate dehydrogenase                    by ATP and ADP
cant activation,         and adenosine has no effect on the reaction.                        It    and the absence of an AMP effect reported by Wieland,                                von Ja-
appears that the phosphate directly attached to the 5’ carbon of                                   gow-Westermann,              and Stukowski          (11) places the mammalian
adenosine may be the preferred group, since ADP and ATP are                                        enzyme in a different category from the microbial                                and higher
less effective than AMP.                No consistent differences between the                      plant keto acid dehydrogenases.




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purine and pyrimidine                 monophosphates             as activators        of the           The effect of nucleotides on the cauliflower KG-dehydrogenase
enzyme are detectable.                                                                             is also different from the E. coli pyruvate                     dehydrogenase            as re-
    It is of interest that the value of the Km for ar-ketoglutarate                           in   ported by Schwartz and Reed (4), who found that this enzyme
the absence of activator,             2.7 X lo-* M, differs slightly from that                     showed relatively            small differences between AMP,                      CMP, and
previously       reported for this enzyme, 1.2 x 10m4 M (6). This                                  GMP in activation                and that it was inhibited                by GTP.          The
appears to be a real and consistent difference which is perhaps                                    cauliflower       KG-dehydrogenase,             which clearly prefers adenylate
attributable       to the change from Tris to N-tris(hydroxymethyl)-                               activators      to other nucleotides,           is strongly activated              by AMP,
methyl-2-aminoethane-sulfonic                    acid buffer          for the standard             less so by ADP; and even less, although                          still significantly          so,
a,ssay used in the present work.                   This difference in K, may be                    by ATP.          These observations           are in accord with those of Shen
related to the relative             buffering effectiveness of the two salts,                      et al. (2), who found that 5 mM ATP in the absence of acetyl-CoA
since Tris is a poor buffer in the region of pH 6.9.                                               resulted in about a 1.5-fold increase in rate as compared with the
    In addition        to the integrated         measure of the activating               effec-    control lacking nucleotide.
tiveness of nucleotides            given by VL,:Kz,               it is possible to ob-                The differing responses to an array of nucleotides,                          when com-
tain from appropriate              experiments       an estimate          of the apparent          pared by means of kinetic parameters                   rather than through obser-
activation      constant (Kl) (8). In the case of coupling activation                              vations of changes in rate at a constant substrate concentration
in which the activator            is capable of binding only to the enzyme-                        brought      about by the same concentration                        of several nucleo-
substrate complex, which appears to apply to the activation                                   of    tides, indicates         that the binding          site for nucleotide             possesses
 KG-dehydrogenase             by nucleotides,       this value is obtained from the                 considerable        specificity     and that, among the nucleotide                     mono-
slope/intercept          of the line of l/Vm,,          against l/[activator].            The       phosphates,        AMP is the best activator.                  Comparisons          of vari-
KL for AMP calculated in this way is 0.025 m&f and that for ADP                                     ously substituted          purine and pyrimidine           nucleotides permit a few
is 0.16 mM, while for ATP the K: is 0.75 mM. These values                                           tentative      conclusions         regarding     the preferred             characteristics,
 cannot be considered as dissociation                 constants for the complex of                  mentioned       above, of the activating            molecule.
 nucleotide with the enzyme-substrate                   complex since, in the treat-                    The question of which component                   of the multienzyme              system
 ment of Friedenwald              and Maengwyn-Davies                   (8), the apparent           used in these studies is the location of the site that binds nucleo-
 dissociation      constant Ki = XK,, where X is a factor expressing                                tide to activate the reaction remains unresolved,                            although       the
 the effect of the activator on the activity                   of the enzyme.          In the       recent work of Davies and Kenworthy                        (12) appears to indicate
 case of coupling activation              of the type seen here, X < 1, indicat-                    that the activating            effect of AMP takes place prior to the de-
 ing that the activator            promotes the association                of enzyme with           carboxylation         of cr-ketoglutarate      in the reaction.            These workers,
 substrate.        Values of X for AMP activation                    of KG-dehydrogen-              measuring        14C02 production          from cY-ketoglutarate              l-14C by an
 ase were calculated by means of a multiple                       regression line fit by            extract of pea mitochondria                obtained       by freezing and thawing
 the method of least squa.res to the equation given by Friedenwald                                  mitochondrial          preparations,       found a 4-fold increase in decar-
 and Maengwyn-Davies                 (8) where :                                                    boxylation       in the presence of 0.5 mM AMP.
                                                                                                        Further     evidence for the tentative              location of the nucleotide
                       v rn%X
                       -=1+-               xK,K,         XK,        XK,                              activating       site in the ar-ketoglutarate-lipoyl                    transsuccinylase
                          V                IS1 [Al + [sl        + [AI                                portion of the complex comes from studies in this laboratory                                 on
                                                                                                     the effect of AMP               on pig heart lipoyl dehydrogenase.                       This
The X value obtained by this means was 0.018, indicating  a strong                                   enzyme, assayed under the same conditions used for the standard
effect of AMP in promoting  the association of the enzyme with its                                  assay for KG-dehydrogenase                  and with reduced lipoic acid as a
substrate.                                                                                           substrate, revealed no consistent effect of the nucleotide.
Issue of March    25, 1971                          R. T. Wedding      and M. K. Bhck                                                    1643

  These observations     appear to point to one of the first two en-     6. POULSEN, L. L., AND WEDDING,      R. T., J. Biol.   Chem.,   246,
zymes in the reaction sequence, but present evidence is inade-           7    5709 (1970).
                                                                           WILKINSON,   G. N., Biochem.   J., 80, 324 (1961).
quate for further localization.                                          8: FRIEDENWALD,   J. S., AND MAENGWYN-DAVIES,      G. D., in W.
                                                                              B. MCELROY AND H. B. GLASS (Editors),       The mechanism
                         REFERENCES                                           of enzyme action, Johns Hopkins   Press, Baltimore,    1954,
                                                                              p. 154.
 1. ATKINSON, D. E., Biochemistry,    7, 4030 (1968).                    9. S&IWARTZ, E. R., AND REED, L. J., Fed. Proc., 27, 389 (1968).
 2. &EN, L. C., FALL, L., WALTON,     G. M., AND ATKINSON,    D. E.,    10. SCHWARTZ. E. R.. OLD. L. 0.. AND REED. L. J.. Biochem.
     Biochemistry, 7, 4041 (1968).                                            Biophws:_ Res. bomm.: 31, 495 (1968).
                                                                                   _                                   ’     ’
3. ATKINSON, D. E., Annu. Rev. Biochem., 36, 85 (1966).                 11. WIELAND, O., VON JAGO&-WESTER&ANN,      B., AND STUKOWSKI,
4. SCHWARTZ, E. R., AND REED, L. J., Biochemistry,      9, 1434               B.. Hovve-Seder’s  2. Phusiol. Chem.. 360. 329 (1969).
     (1970).                                                            12. DA&ES, K. D.,AND KENWORTHY, P., J.‘Esp.. Botaky, 2i, 247
5. REED, L. J., J. Vitaminol.    (Osaka),   14, 77 (1968).                    (1970).




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