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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 Downloaded from www.jbc.org by guest, on December 9, 2011 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 Downloaded from www.jbc.org by guest, on December 9, 2011 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 Downloaded from www.jbc.org by guest, on December 9, 2011 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 Downloaded from www.jbc.org by guest, on December 9, 2011 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. Downloaded from www.jbc.org by guest, on December 9, 2011 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). Downloaded from www.jbc.org by guest, on December 9, 2011
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