scholarly journals Interaction between the individual isoenzymes of pyruvate dehydrogenase kinase and the inner lipoyl-bearing domain of transacetylase component of pyruvate dehydrogenase complex

2002 ◽  
Vol 366 (1) ◽  
pp. 129-136 ◽  
Author(s):  
Alina TUGANOVA ◽  
Igor BOULATNIKOV ◽  
Kirill M. POPOV
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Kinase Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Tight Binding ◽  
Enzymic Activity ◽  
Pyruvate Dehydrogenase Kinase ◽  
Size Exclusion ◽  
Apparent Dissociation Constant ◽  
The Individual ◽  
Dehydrogenase Complex

Protein—protein interactions play an important role in the regulation of enzymic activity of pyruvate dehydrogenase kinase (PDK). It is generally believed that the binding of PDK to the inner lipoyl-bearing domain L2 of the transacetylase component E2 of pyruvate dehydrogenase complex largely determines the level of kinase activity. In the present study, we characterized the interaction between the individual isoenzymes of PDK (PDK1—PDK4) and monomeric L2 domain of human E2, as well as the effect of this interaction on kinase activity. It was found that PDK isoenzymes are markedly different with respect to their affinities for L2. PDK3 demonstrated a very tight binding, which persisted during isolation of PDK3—L2 complexes using size-exclusion chromatography. Binding of PDK1 and PDK2 was readily reversible with the apparent dissociation constant of approx. 10μM for both isoenzymes. PDK4 had a greatly reduced capacity for L2 binding (relative order PDK3>PDK1 = PDK2>PDK4). Monomeric L2 domain alone had very little effect on the activities of either PDK1 or PDK2. In contrast, L2 caused a 3-fold increase in PDK3 activity and approx. 37% increase in PDK4 activity. These results strongly suggest that the interactions between the individual isoenzymes of PDK and L2 domain are isoenzyme-specific and might be among the major factors that determine the level of kinase activity of particular isoenzyme towards the pyruvate dehydrogenase complex.

Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites

2001 ◽  
Vol 358 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Elena KOLOBOVA ◽  
Alina TUGANOVA ◽  
Igor BOULATNIKOV ◽  
Kirill M. POPOV
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Phosphorylation Site ◽  
Enzymic Activity ◽  
Mammalian Tissues ◽  
Activity State ◽  
The Individual ◽  
Pyruvate Dehydrogenase Phosphatase ◽  
Thiamin Pyrophosphate ◽  
Dehydrogenase Complex

The enzymic activity of the mammalian pyruvate dehydrogenase complex is regulated by the phosphorylation of three serine residues (sites 1, 2 and 3) located on the E1 component of the complex. Here we report that the four isoenzymes of protein kinase responsible for the phosphorylation and inactivation of pyruvate dehydrogenase (PDK1, PDK2, PDK3 and PDK4) differ in their abilities to phosphorylate the enzyme. PDK1 can phosphorylate all three sites, whereas PDK2, PDK3 and PDK4 each phosphorylate only site 1 and site 2. Although PDK2 phosphorylates site 1 and 2, it incorporates less phosphate in site 2 than PDK3 or PDK4. As a result, the amount of phosphate incorporated by each isoenzyme decreases in the order PDK1>PDK3PDK4>PDK2. Significantly, binding of the coenzyme thiamin pyrophosphate to pyruvate dehydrogenase alters the rates and stoichiometries of phosphorylation of the individual sites. First, the rate of phosphorylation of site 1 by all isoenzymes of kinase is decreased. Secondly, thiamin pyrophosphate markedly decreases the amount of phosphate that PDK1 incorporates in sites 2 and 3 and that PDK2 incorporates in site 2. In contrast, the coenzyme does not significantly affect the total amount of phosphate incorporated in site 2 by PDK3 and PDK4, but instead decreases the rate of phosphorylation of this site. Furthermore, pyruvate dehydrogenase complex phosphorylated by the individual isoenzymes of kinase is reactivated at different rates by pyruvate dehydrogenase phosphatase. Both isoenzymes of phosphatase (PDP1 and PDP2) readily reactivate the complex phosphorylated by PDK2. When pyruvate dehydrogenase is phosphorylated by other isoenzymes, the rates of reactivation decrease in the order PDK4PDK3> PDK1. Taken together, results reported here strongly suggest that the major determinants of the activity state of pyruvate dehydrogenase in mammalian tissues include the phosphorylation site specificity of isoenzymes of kinase in addition to the absolute amounts of kinase and phosphatase protein expressed in mitochondria.

scholarly journals Long-term regulation of pyruvate dehydrogenase complex. Evidence that kinase-activator protein (KAP) is free pyruvate dehydrogenase kinase

1991 ◽  
Vol 275 (3) ◽  
pp. 781-784 ◽  
Author(s):  
B S Jones ◽  
S J Yeaman
Keyword(s):  
Pyruvate Dehydrogenase ◽  
High Speed ◽  
Kinase Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Phosphorylation Site ◽  
Pyruvate Dehydrogenase Kinase ◽  
Activator Protein ◽  
Major Phosphorylation Site ◽  
Dehydrogenase Complex

The kinase-activator protein (KAP) of pyruvate dehydrogenase complex (PDC) has been purified approx. 2250-fold from high-speed supernatants of mitochondrial extracts from the liver of 48 h-starved rats. Purified KAP demonstrates kinase activity towards both the E1 component of PDC and towards a synthetic peptide corresponding to the major phosphorylation site on E1. Furthermore, the activities of KAP and PDC kinase co-fractionate through several stages of purification and have the same apparent mass. We conclude that KAP is not a distinct protein, but is kinase which has dissociated from the complex.

scholarly journals Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex

1998 ◽  
Vol 329 (1) ◽  
pp. 191-196 ◽  
Author(s):  
Melissa M. BOWKER-KINLEY ◽  
I. Wilhelmina DAVIS ◽  
Pengfei WU ◽  
A. Robert HARRIS ◽  
M. Kirill POPOV
Keyword(s):  
Tissue Distribution ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Dehydrogenase Kinase ◽  
Synthetic Analogue ◽  
Complex Activity ◽  
Acetyl Coa ◽  
Tissue Specific ◽  
Specific Regulation ◽  
Dehydrogenase Complex

Tissue distribution and kinetic parameters for the four isoenzymes of pyruvate dehydrogenase kinase (PDK1, PDK2, PDK3 and PDK4) identified thus far in mammals were analysed. It appeared that expression of these isoenzymes occurs in a tissue-specific manner. The mRNA for isoenzyme PDK1 was found almost exclusively in rat heart. The mRNA for PDK3 was most abundantly expressed in rat testis. The message for PDK2 was present in all tissues tested but the level was low in spleen and lung. The mRNA for PDK4 was predominantly expressed in skeletal muscle and heart. The specific activities of the isoenzymes varied 25-fold, from 50 nmol/min per mg for PDK2 to 1250 nmol/min per mg for PDK3. Apparent Ki values of the isoenzymes for the synthetic analogue of pyruvate, dichloroacetate, varied 40-fold, from 0.2 mM for PDK2 to 8 mM for PDK3. The isoenzymes were also different with respect to their ability to respond to NADH and NADH plus acetyl-CoA. NADH alone stimulated the activities of PDK1 and PDK2 by 20 and 30% respectively. NADH plus acetyl-CoA activated these isoenzymes nearly 200 and 300%. Under comparable conditions, isoenzyme PDK3 was almost completely unresponsive to NADH, and NADH plus acetyl-CoA caused inhibition rather than activation. Isoenzyme PDK4 was activated almost 2-fold by NADH, but NADH plus acetyl-CoA did not activate above the level seen with NADH alone. These results provide the first evidence that the unique tissue distribution and kinetic characteristics of the isoenzymes of PDK are among the major factors responsible for tissue-specific regulation of the pyruvate dehydrogenase complex activity.

Increased pyruvate dehydrogenase kinase activity in response to sepsis

1991 ◽  
Vol 260 (5) ◽  
pp. E669-E674 ◽  
Author(s):  
T. C. Vary
Keyword(s):  
Skeletal Muscle ◽  
Pyruvate Dehydrogenase ◽  
Kinase Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Dehydrogenase Kinase ◽  
Sterile Inflammation ◽  
Stable Factor ◽  
Skeletal Muscle Mitochondria ◽  
Dependent Inactivation ◽  
Significant Difference

The effect of sterile inflammation and sepsis on the proportion of active pyruvate dehydrogenase complex (PDH) in mitochondria isolated from skeletal muscle has been investigated. The proportion of active PDH in mitochondria isolated from septic animals was significantly reduced compared with control under all incubation conditions examined, even in the presence of inhibitors of the PDH kinase. There was no significant difference between control and sterile inflammation in any of the incubations examined. The rate constant for ATP-dependent inactivation of the PDH complex in mitochondrial extracts from control animals was -0.42 min-1 (r = 0.993; P less than 0.001) and was not altered in mitochondrial extracts from sterile inflammatory animals (-0.43 min-1; r = 0.999; P less than 0.001). However, rate constants for inactivation in septic animals was significantly increased over twofold to -1.08 min-1 (r = 0.987; P less than 0.001) (P less than 0.001 vs. control or sterile inflammation). In the presence of inhibitors of the PDH kinase reaction (2.5 mM pyruvate or 1 mM dichloroacetate), inactivation of PDH after addition of ATP was significantly greater in mitochondrial extracts from septic than either control or sterile inflammatory animals. These results suggest that sepsis, but not sterile inflammation, induces a stable factor in skeletal muscle mitochondria that increased PDH kinase activity.

scholarly journals The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

2020 ◽  
Author(s):  
Jaehyoun Lee ◽  
Seunghee Oh ◽  
Saikat Bhattacharya ◽  
Ying Zhang ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Pyruvate Dehydrogenase ◽  
Biochemical Analysis ◽  
Pyruvate Dehydrogenase Complex ◽  
Size Exclusion ◽  
Dependent Manner ◽  
Acetyl Coa ◽  
Cell Extracts ◽  
Exclusion Chromatography ◽  
Dehydrogenase Complex

ABSTRACTThe pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.

Abstract TP268: Combination Therapy of Normobaric Oxygen With Hypothermia or Ethanol Modulates Pyruvate Dehydrogenase Complex in Thromboembolic Cerebral Ischemia

Stroke ◽  
2016 ◽  
Vol 47 (suppl_1) ◽  
Author(s):  
Alexa Thibodeau ◽  
Lipeng Cai ◽  
Changya Peng ◽  
Xiaokun Geng ◽  
Vicki Diaz ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Protein Expression ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Pyruvate Dehydrogenase Kinase ◽  
Tissue Type ◽  
Energy Regulation ◽  
Normobaric Oxygen ◽  
Expression Levels ◽  
Dehydrogenase Complex

Background and Hypothesis: Pyruvate Dehydrogenase Complex (PDH) is a brain mitochondrial matrix enzyme that is inactivated during stroke injury. PDH impairment after stroke can be particularly devastating given PDH’s critical role in the conversion from anaerobic to aerobic energy metabolism. In this study, we evaluated the restoration of oxidative metabolism by measuring reactive oxygen species (ROS) levels and energy regulation by characterizing modulation of PDH and its inhibitor, pyruvate dehydrogenase kinase (PDK), with therapeutic combination of normobaric oxygen (NBO) plus either hypothermia (Hypo) or ethanol (EtOH). Methods: Sprague-Dawley rats were subjected to middle cerebral artery (MCA) occlusion induced with an autologous embolus, the more clinically relevant stroke model. One hour after occlusion, tissue-type plasminogen activator (t-PA) was administered alone or with NBO (60%), EtOH (1.0g/kg) or Hypo (33°C), either singly or in combination. PDH activity and ROS levels were measured at 3 and 24 hours after t-PA administration. Western blotting was used to detect PDH and PDK protein expression levels. Results: Administration of 60% NBO alone after reperfusion by t-PA treatment did not affect PDH activity. Under t-PA, compared to EtOH or Hypo alone, combined administration of NBO plus either EtOH or Hypo produced the greatest increases in PDH activity and protein expression levels, as well as the greatest decrease in PDK expression. Combination therapy also provided the most significant decline in ROS generation compared to any monotherapeutic approach. Conclusions: Reperfusion with t-PA followed by 60% NBO improves the efficacy of EtOH or Hypo in neuroprotection by ameliorating oxidative injury and improving metabolic regulation with PDH. Comparable neuroprotective effects were found when treating with either EtOH or Hypo, suggesting a similar mechanism and the possibility of substituting EtOH for Hypo in the clinical setting.

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