A unifying mechanism for stimulation of mammalian pyruvate dehydrogenase(a) kinase by reduced nicotinamide adenine dinucleotide, dihydrolipoamide, acetyl coenzyme A, or pyruvate.

Marked increases in the rates of phosphorylation and inactivation of the pyruvate dehydrogenase complex by pyruvate dehydrogenase, (PDH,) kinase can be induced by NADH, acetyl-coenzyme A or, under certain conditions, pyruvate. Our results support the hypothesis that reduction (by NADH) or acetylation (by acetyl-CoA or pyruvate) of the lipoyl moiety of the dihydrolipoyl transacetylase component of the complex is required for stimulation of the PDH, kinase activity by these effecters. NADH and free dihydrolipoamide give half-maximal stimulation of PDH, kinase activity at about 4 and 7 PM, respectively. Consistent with a common mechanism for stimulation by these effecters, a combination of 50 PM NADH and 50 FM dihydrolipoamide gives no additional stimulation beyond either added individually at the same concentration. At a concentration of 50 PM, NADH or dihydrolipoamide facilitates acetyl-CoA stimulation, which does not occur in the absence of a reducing compound. At the same thiol concentration, dithiothreitol, dithioerythritol, or /3-mercaptoethanol fail either to stimulate PDH, kinase activity or to facilitate acetyl-CoA stimulation. Dihydrolipoamide (50 FM) stimulates PDH, kinase activity when added in addition to a IO-fold higher concentration of these other thiol compounds. Thus, the effects of dihydrolipoamide on PDH, kinase activity are very specific and closely parallel NADH stimulation and NADH-facilitated acetylCoA stimulation of PDH, kinase activity. Depending on conditions, pyruvate either stimulates or inhibits PDH, kinase activity. Acetyl-CoA and low pyruvate concentrations stimulate PDH, kinase activity to about the same level and the combination of pyruvate an.d acetyl-CoA gives no additional stimulation. Acetyl-CoA and pyruvate have parallel salt dependence with greater than 20 mM KC1 required to detect stimulation by either. These results are consistent with a common mechanism for stimulation of

Marked increases in the rates of phosphorylation and inactivation of the pyruvate dehydrogenase complex by pyruvate dehydrogenase, (PDH,) kinase can be induced by NADH, acetyl-coenzyme A or, under certain conditions, pyruvate. Our results support the hypothesis that reduction (by NADH) or acetylation (by acetyl-CoA or pyruvate) of the lipoyl moiety of the dihydrolipoyl transacetylase component of the complex is required for stimulation of the PDH, kinase activity by these effecters.
NADH and free dihydrolipoamide give half-maximal stimulation of PDH, kinase activity at about 4 and 7 PM, respectively.
Consistent with a common mechanism for stimulation by these effecters, a combination of 50 PM NADH and 50 FM dihydrolipoamide gives no additional stimulation beyond either added individually at the same concentration.
At a concentration of 50 PM, NADH or dihydrolipoamide facilitates acetyl-CoA stimulation, which does not occur in the absence of a reducing compound. At the same thiol concentration, dithiothreitol, dithioerythritol, or /3-mercaptoethanol fail either to stimulate PDH, kinase activity or to facilitate acetyl-CoA stimulation.
Dihydrolipoamide (50 FM) stimulates PDH, kinase activity when added in addition to a IO-fold higher concentration of these other thiol compounds. Thus, the effects of dihydrolipoamide on PDH, kinase activity are very specific and closely parallel NADH stimulation and NADH-facilitated acetyl-CoA stimulation of PDH, kinase activity. Depending on conditions, pyruvate either stimulates or inhibits PDH, kinase activity. Acetyl-CoA and low pyruvate concentrations stimulate PDH, kinase activity to about the same level and the combination of pyruvate an.d acetyl-CoA gives no additional stimulation. Acetyl-CoA and pyruvate have parallel salt dependence with greater than 20 mM KC1 required to detect stimulation by either. These results are consistent with a common mechanism for stimulation of PDH, kinase activity by acetyl-CoA and pyruvate. Phosphate anion, which itself lowers PDH, kinase activity, enhances inhibition of PDH, kinase activity by high concentrations of pyruvate. However, phosphate anion does not abolish pyruvate stimulation of PDH, kinase activity, which is still observed with low pyruvate concentrations and parallels acetyl-CoA stimulation.
Pyruvate dehydrogenase complex, containing acetylated dihydrolipoyl moieties, was prepared by treatment with [3-Wlpyruvate followed by gel filtration. Stimulation of PDH, kinase persists with gel filtered enzyme and addition of pyruvate gives no additional stimulation. Furthermore, addition of acetyl-CoA, NADH or a combination of acetyt-CoA and NADH gives little or no additional stimulation of PDH, kinase activity. These results further support the proposed involvement of the lipoyl moieties since acetylation of lipoyl moieties is associated with enhanced PDH, kinase activity and elimination of stimulation by pyruvate, acetyl-CoA, and NADH.
The transition state analog, thiamin thiazolone pyrophosphate, blocks acetylation of the lipoyl moieties by [3-Wlpyruvate and prevents pyruvate stimulation of PDH, kinase activity. However, the analog, although an inhibitor of PDH, kinase activity, does not prevent stimulation of PDH, kinase activity by NADH or acetyl-CoA. Other

RESULTS
Acetyl-CoA failed to stimulate PDH, kinase in the absence of an added thiol or other reductant such as NADH. Low concentrations of either NADH or dihydrolipoamide (50 PM) facilitated a high level of acetyl-CoA stimulation beyond that due to dihydrolipoamide (or NADH) alone. However, 50 PM dithiothreitol or dithioerythritol or 100 pM /3-mercaptoethanol did not allow significant stimulation of PDH, kinase by acetyl-CoA. At a concentration of 2 mM, dithiothreitol and, to a lesser extent, dithioerythritol were effective in facilitating acetyl-CoA stimulation (Table I) In contrast to dihydrolipoamide, which gave maximal stimulation of PDH, kinase activity at 50 pM, dithiothreitol or dithioerythritol at 50 PM or P-mercaptoethanol at 100 pM not only failed to stimulate, but slightly inhibited, PDH, kinase activity (Table I). As previously observed (141, 2 mM dithiothreitol gave slight stimulation of PDH, kinase activity. However, addition of 50 PM dihydrolipoamide on top of 2 mM dithiothreitol gave much higher levels of stimulation. Similar pronounced increases were observed with 50 PM dihydrolipoamide on top of 2 mM dithioerythritol and 5 mM P-mercaptoethanol (Table I). These data clearly support the conclusion that stimulation by dihydrolipoamide results from a highly specific interaction rather than a general thiol effect.
nol, at a concentration of 5 mM, did not allow acetyl-CoA stimulation. These results further support the high specificity of dihydrolipoamide and its similarity to NADH. The high level of acetyl-CoA stimulation observed in the presence of dithiothreitol is consistent with our previous observation that, at high concentrations, dithiothreitol can serve as a source of reducing power for the catalytic reduction of NAD+ by the pyruvate dehydrogenase complex (14).  Fig. 3 discussed below). Since ADP is a more effective inhibitor of PDH, kinase at higher levels of KC1 (7), the stimulations observed at high KC1 concentrations might be at least partially due to reduction of ADP inhibition. The degree of stimulation of PDH, kinase activity was increased with ADP inhibited enzyme. However, at 60 mM KCl, pyruvate, NADH, and acetyl-CoA did not change either the Michaelis constant for ATP or the competitive inhibition constant for ADP with respect to ATP.
Most of the assays of PDH, kinase activity in the present paper were conducted with an ADP:ATP ratio of 5:l. Our pyruvate dehydrogenase complex preparations have usually contained high levels of endogenous PDH, kinase activity. ADP inhibition of PDH, kinase improves our ability to make initial velocity measurements with low depletion (i.e. phosphorylation) of the protein substrate. For the above kinetic studies, a particular preparation with lower than normal PDH, kinase activity was employed. same level (Table II). The combination of pyruvate and acetyl-CoA did not yield any additional stimulation. The absence of any additivity clearly supports the premise that acetyl-CoA and pyruvate stimulate PDH, kinase activity by the same mechanism. The results described below also support this conclusion.   Fig. 3, the control values decrease with increasing KC1 primarily due to increased ADP inhibition (7) but also due to a slight decrease in kinase activity at higher KC1 concentrations (i.e. in the absence of ADP). The results in Fig. 3 reveal a close parallel between pyruvate and acetyl-CoA stimulation; with either effector, KC1 concentrations greater than 20 mM were required before significant stimulation was observed. Both effecters were inhibitory in the absence of KCl. The lower level of stimulation achieved with 2 mM pyruvate than with 0.1 mM pyruvate presumably reflects some pyruvate inhibition of PDH, kinase activity at the higher concentration.
The results, shown in Fig. 4 should persist following removal of pyruvate. Acetylated pyruvate dehydrogenase complex was prepared by treatment with 0.25 mM [3-14Clpyruvate followed by gel filtration in the presence of a buffer containing 10 pM TPP. Removal of virtually all of the pyruvate by gel filtration was confirmed by a parallel experiment in which [1-iV]pyruvate was used. Control pyruvate dehydrogenase complex also underwent gel filtration and the protein concentrations of the gel-filtered enzymes were measured. There was no change in the level of acetylation of the [3-'Clpyruvate-treated enzyme from the beginning to the end of the PDH, kinase assays. An important condition for achieving this result was conducting the studies in the absence of dithiothreitol.
The results, in Fig. 5, show that stimulation due to pyruvate treatment persisted after removal of pyruvate. Addition of 0.1 mM pyruvate to the gel-filtered, acetylated pyruvate dehydrogenase complex not only failed to cause further stimulation but slightly inhibited PDH, kinase activity. These results support the hypothesis that pyruvate stimulation results from formation of acetylated lipoyl moieties.
It is also possible that pyruvate stimulation could result from removal of TPP inhibition by formation of a-hydroxyethyl thiamin pyrophosphate (HETPP). However, since gel filtration was conducted in the presence of 10 pM TPP, a significant rate of exchange of TPP with HETPP should have removed the stimulatory effect as explained by this postulate. in TPP -Acetylated pyruvate dehydrogenase complex was prepared as described above except that 12 PM TPP was present with the pyruvate dehydrogenase complex (at 12 mglml) during acetylation with [3-14C1pyruvate. Gel filtration was conducted in the absence of thiamin pyrophosphate. Following gel filtration, the acetylated enzyme was adjusted to 2 mg/ml. It was assumed that all the TPP (primarily as tightly bound HETPP3) remained with the pyruvate-treated complex and was now present at a concentration of 2 PM. That level of TPP was added to the control enzyme.
In Table III comparisons of PDH, kinase activities are presented both as absolute rates for comparing the activities associated with acetylated or nonacetylated pyruvate dehydrogenase complexes and as per cent activities for comparison of the effects of compounds added. As described previously, addition of pyruvate did not cause additional stimulation of PDH, kinase activity associated with acetylated pyruvate dehydrogenase complex. Addition of pyruvate to the control complex caused stimulation of PDH, kinase activity to approximately the same level as the PDH, kinase activity associated with the acetylated complex. There was little, if any, decrease in the level of protein-bound acetyl groups in the acetylated complex due to this treatment.
For the TPP-treated complexes, comparison of the activity of the PDH, kinase reveals that, although inhibited, the activity associated with the acetylated complex is much higher than the activity associated with the nonacetylated complex. Furthermore, contrary to the proposal that pyruvate stimulates by releasing TPP inhibition, pyruvate caused no significant increase in PDH, kinase activity associated with the acetylated and TPP-treated enzyme complex. There was a small increase in the level of protein-bound acetyl groups which may contribute to the slight stimulation observed. As described above, pyruvate caused a large increase in the activity of PDH, kinase associated with the control enzyme. It is not clear why the PDH, kinase activity associated with both forms of the complex did not have the same level of activity following addition of pyruvate and TPP. Neither of the proposed mechanisms predicts this difference.
Whereas acetyl-CoA (plus dithiothreitol), NADH, and NADH plus acetyl-CoA caused additional stimulation of PDH, kinase activity associated with the nonacetylated complex, these effecters gave little or no stimulation of the PDH, kinase activity associated with the acetylated complex (Table  III). This result clearly supports the hypothesis that formation of acetylated lipoates saturated the sites for stimulation by these effecters.
Coenzyme A inhibited the activity of PDH, kinase associated with both the acetyl-ated and nonacetylated complex. Since CoA treatment of acetylated pyruvate dehydrogenase complex would result in formation of the reduced form of the lipoyl moiety, our model predicts a higher level of PDH, kinase activity associated with this form of the complex than with the nonacetylated but CoA-treated control. This was observed (Table III).

Effects
of Thiamin Thiazolone Pyrophosphate on Pyruvate and Acetyl-CoA Stimulations ofPDH, Kinase -Thiamin thiazolone pyrophosphate was shown by Gutowski and Lienhard (19) to have properties expected for a transition state analog.  (20).
The following studies with thiamin thiazolone pyrophosphate aided in showing that acetylation of the lipoyl moieties was required for pyruvate stimulation of PDH, kinase activity. In Table IV, the activity of PDH, kinase and the level of acetylation of lipoyl moieties were compared for a preparation of kidney pyruvate dehydrogenase complex that was dependent on added TPP (5% activity in the absence of added TPP) and this enzyme to which TPP or TTPP had been added. The latter enzyme was incubated with TTPP until it was virtually completely inactive in the overall reaction catalyzed by the pyruvate dehydrogenase complex.
Both TPP and TTPP inhibited PDH, kinase activity (Table  IV). Assuming TTPP binds at the same site as HETPP, this result and the results described in the previous section are consistent with HETPP causing inhibition of PDH, kinase activity. With the TPP-treated enzyme, there was an increase in both the level of protein-bound acetyl groups and pyruvate stimulation of PDH, kinase activity. In contrast, TTPP prevented acetylation of lipoyl moieties and eliminated pyruvate stimulation. These findings further support the proposal that pyruvate stimulation of PDH, kinase activity requires acetylation of bound lipoyl moiety.
In other studies with TTPP-treated pyruvate dehydrogenase complex (not shown), acetyl-CoA and NADH stimulations of PDH, kinase activity were observed. Also phosphate-enhanced pyruvate inhibition of PDH, kinase activity was observed.

DISCUSSION
Our results demonstrate a close parallel between NADH and dihydrolipoamide and between acetyl-CoA and pyruvate in stimulating PDH, kinase activity. These comparisons reveal similar levels of stimulation and a complete lack of additivity. Significantly higher levels of PDH, kinase activity are achieved with acetyl-CoA or pyruvate than with NADH or dihydrolipoamide.
This result suggests that these pairs stimulate PDH, kinase activity by different mechanisms. A common mechanism, such as stimulation of PDH, kinase Stimulation of Mammalian Pyruvate Dehydrogenase, Kinase activity resulting from release of inhibition by the oxidized form of the lipoyl moiety (21), seems unlikely. According to our proposed mechanism (141, illustrated in Fig. 6, the acetylated form of the lipoyl moiety would be more effective than the reduced form in stimulating PDH, kinase activity. Our results clearly indicate that the stimulation of PDH, kinase activity by free dihydrolipoamide does not result from a general thiol effect but is highly specific. At the same concentrations, other thiol compounds could not replace dihydrolipoamide in stimulating PDH, kinase or facilitating acetyl-CoA stimulation.
In addition, dihydrolipoamide could stimulate PDH, kinase activity in the presence of much higher concentrations of other thiol compounds.
At the concentration that gives half-maximal stimulation, dihydrolipoamide is present at a slightly lower concentration than the subunits of dihydrolipoyl transacetylase and, therefore, lower than the stoichiometry of protein-bound lipoyl moieties (17). Thus it seems most likely that the free dihydrolipoamide directly stimulates PDH, kinase activity. However, the catalytic reduction of the covalently bound lipoyl moiety might occur following the reduction first at the active site of the dihydrolipoyl dehydrogenase component by the free dihydrolipoamide.
Even lower concentrations of NADH (and, therefore, stoichiometries with respect to bound lipoyl moieties) stimulate PDH, kinase activity.
As indicated in the introductory statement, pyruvate stimulation of PDH, kinase activity probably is not an important factor in the modulation of this regulatory enzyme in uiuo. However, among the evidence supporting an indirect mechanism for stimulation of PDH, kinase activity, studies with pyruvate are of central importance. Our studies show that higher PDH, kinase activity persists following treatment with pyruvate and gel filtration to remove free pyruvate. An important observation is that NADH and acetyl-CoA can no longer stimulate PDH, kinase activity associated with acetylated pyruvate dehydrogenase complex. This is consistent with the concept that NADH and acetyl-CoA cannot stimulate PDH, kinase activity because stimulation occurs by the reduction and acetylation of lipoyl moieties and this effect has been saturated by acetylation of the lipoyl moieties by pyruvate.
TPP inhibits PDH, kinase activity (5). A possible explanation of pyruvate stimulation of PDH, kinase is that formation of the active aldehyde intermediate following decarboxylation of pyruvate might release this inhibition. This proposal is not consistent with the observation that with acetylated pyruvate dehydrogenase complex, deficient in TPP, addition of TPP inhibits PDH, kinase activity and addition of pyruvate does not reverse this inhibition.
Since under some conditions pyruvate inhibits PDH, kinase activity, it was important to select conditions for studying pyruvate stimulation that minimize pyruvate inhibition. Our studies involving variation in the levels of KC1 and potassium phosphate revealed not only a close parallel in the salt dependence for stimulation of PDH, kinase activity by pyruvate and acetyl-CoA but also enhancement by phosphate anion of pyruvate inhibition of PDH, kinase activity. Dichloroacetate also inhibits PDH, kinase activity (22). In Mops/ sodium buffer, inhibition by dichloroacetate is also increased by increasing potassium phosphate in the range of 2 to 20 mM.4 This suggests that pyruvate and dichloroacetate may inhibit PDH, kinase activity by binding at the same site. In previous studies (4) of pyruvate inhibition of PDH, kinase activity, the inhibition constant for pyruvate was observed to be somewhat variable. This may be a consequence of a mixture of stimulation and inhibition of PDH, kinase activity causing variation in the apparent inhibition constant. PDH, kinase has a low molecular activity, -15 mol of 32P incorporated/min/mol of PDH, kinase. For the overall reaction catalyzed by the pyruvate dehydrogenase complex, there is a molecular activity of about 175 mol/min/mol of subunit of the dihydrolipoyl transacetylase.
Thus for each turnover of the PDH, kinase, the lipoyl moieties of the transacetylase undergo several transformations between the oxidized, reduced, and acetylated forms. Since PDH, kinase activity is much slower than interconversion of the lipoyl moieties, there is an increased probability of association of a stimulatory form between catalytic turnovers by PDH, kinase. It is also possible that unique and specific lipoyl moieties may be associated with PDH, kinase that can undergo reduction and acetylation and that these have a particularly high probability of interacting with PDH, kinase. Thus our proposed mechanism does not require the interaction of the reduced and acetylated forms of the lipoyl moiety at a site causing stimulation of PDH, kinase activity to be directly proportional to the affinity of substrates and products. Recently we observed that very low NADH:NAD+ ratios and acetyl-CoA:CoA ratios effectively reduce the activity of liver or kidney pyruvate dehydrogenase complex by enhancing PDH, kinase activity while much higher ratios are required for product inhibition of the overall reaction catalyzed by the complex (16).
In the process of resolving the pyruvate dehydrogenase complex into its component enzymes, the PDH, kinase tends to remain tightly associated with the core dihydrolipoyl transacetylase component (23). Based on binding studies with [WIATP, there appear to be about 2 to 4 PDH, kinase molecules associated with our preparations of kidney pyruvate Pyruvate Dehydrogenase, Kinase 503 dehydrogenase complex.5 In addition to our observation that very low ratios of NADH or dihydrolipoamide to bound lipoyl moieties can stimulate PDH, kinase activity, we have observed that PDH, kinase activity increases appreciably with the level of acetylation at lower levels of acetylation but is increased to a lesser degree by higher levels of acetylation. These observations, together with consideration of the low stoichiometry of PDH, kinase associated with the pyruvate dehydrogenase complex, are consistent with the possibility that PDH, kinase can be maximally stimulated by reduction or acetylation of a few or possibly specific dihydrolipoyl residues.
An interesting possibility is that PDH, kinase is associated with a specific and unique subunit of the dihydrolipoyl transacetylase core that contains a lipoyl moiety and that this subunit functions as a regulatory subunit of the PDH, kinase. This model would require the pyruvate dehydrogenase components to undergo phosphorylation by moving to the site of the fixed PDH, kinase. We have prepared highly phosphorylated (completely inactive) pyruvate dehydrogenase complex which allows only a low level of acetylation of lipoyl moieties. After mixing this with nonphosphorylated complex for 15 s and then adding pyruvate, there was a pronounced increase in protein-bound acetyl groups (i.e. beyond the additive levels measured with phosphorylated and nonphosphorylated complex assayed individually).
This indicates that the pyruvate dehydrogenase components can exchange from one dihydrolipoyl transacetylase core to another.
In conclusion, our data support a regulatory mechanism involving the following sequence: (a) substrates and products of the overall reaction bind and undergo conversion at topographically distinct catalytic sites; (b) these diverse catalytic events alter the distribution of the covalently bound lipoyl moiety between the oxidized, acetylated, and reduced forms; and (c) this changes the degree of interaction by the acetylated and reduced forms of this mobile carrier at a specific site (or sites), thereby modifying PDH, kinase activity.