Notice: Wiley Online Library will be unavailable on Saturday 27th February from 09:00-14:00 GMT / 04:00-09:00 EST / 17:00-22:00 SGT for essential maintenance. Apologies for the inconvenience.
T. E. Roche, Department of Biochemistry, Willard Hall, Kansas State University, Manhattan, KS 66506, USA. Fax: +1 785 532 7278, Tel.: + 1 785 532 6116, E-mail: firstname.lastname@example.org
Four pyruvate dehydrogenase kinase and two pyruvate dehydrogenase phosphatase isoforms function in adjusting the activation state of the pyruvate dehydrogenase complex (PDC) through determining the fraction of active (nonphosphorylated) pyruvate dehydrogenase component. Necessary adaptations of PDC activity with varying metabolic requirements in different tissues and cell types are met by the selective expression and pronounced variation in the inherent functional properties and effector sensitivities of these regulatory enzymes. This review emphasizes how the foremost changes in the kinase and phosphatase activities issue from the dynamic, effector–modified interactions of these regulatory enzymes with the flexibly held outer domains of the core-forming dihydrolipoyl acetyl transferase component.
If you can't find a tool you're looking for, please click the link at the top of the page to "Go to old article view". Alternatively, view our Knowledge Base articles for additional help. Your feedback is important to us, so please let us know if you have comments or ideas for improvement.
The mitochondrial pyruvate dehydrogenase complex (PDC) plays a critical fuel selection role in determining whether glucose-linked substrates are converted to acetyl-CoA [1–4]. When carbohydrate stores are reduced, mammalian PDC activity is down-regulated and limits the oxidative utilization of glucose in most non-neural tissues. Extended starvation results in PDC activity being profoundly suppressed in most tissues; operation of the same regulatory control severely restricts PDC activity in diabetic animals to thereby impede consumption of abundant glucose. Following the intake of excess dietary carbohydrate, activation of PDC in fat synthesizing tissues accelerates fatty acid biosynthesis from glucose. Adaptable control of PDC activity is required to satisfy these diverse tasks in the management of fuel consumption and storage. This is achieved by the tissue-specific and metabolic state-specific expression and the discrete regulatory properties of the dedicated kinase and phosphatase isozymes [1–4]. Four pyruvate dehydrogenase kinase (PDK) isozymes and two pyruvate dehydrogenase phosphatase (PDP) isoforms function in governing the activity state of PDC [5–7]. In combination these carry out a continuous interconversion cycle that determines the proportion of the pyruvate dehydrogenase (E1) component that is in the active, nonphosphorylated state.
Among the regulatory enzymes only PDP isoform 1 (PDP1) was purified and its distinct regulatory properties characterized  prior to the relatively recent development of the capacity to recombinantly express the kinase and phosphatase isoforms. Although prior studies established a set of prototypical regulatory responses for kinase function, these were ascertained from studies on purified complexes and resolved kinase fractions containing an undefined mixture of kinase isoforms. PDK isozymes together with the related branched-chain dehydrogenase kinase constitute a novel family of serine kinases, unrelated to cytoplasmic Ser/Thr/Tyr kinases [1,2,5,6,9–12]. Based on the order in which they were initially cloned, the four PDK isoforms identified in mammals are designated PDK1, PDK2, PDK3 and PDK4. Though not related to the cytoplasmic Ser/Thr/Tyr kinases, these PDK isoforms have a 2-domain structure with a C-terminal domain that is related to another class of ATP consuming enzymes [9–12] that broadly includes bacterial histidine kinases. As detailed elsewhere , sequence comparisons of the same PDK isozyme from different mammals are highly conserved for each of the four isoforms (> 94% sequence identity for human vs. rat). Comparison of any combination of the different 45.5–46 kDa human isoforms reveals that they share 65% ± 4% sequence identity, with only short segments at the N-terminus that cannot be aligned.
The two PDP isoforms have catalytic subunits that are members of the 2C class of protein phosphatases [7,13]. Both PDP activities require Mg2+ and are regulated with regard to their responsiveness to this essential metal [7,8]. Micromolar Ca2+ greatly stimulates the activity of PDP1. Polyamines, most effectively spermine, markedly reduce the Km values for Mg2+ of both PDP isoforms. The Km of PDP2 in the absence of polyamines is very high (16 mm) and reduced to 3 mm; whereas the Km of PDP1 for Mg2+ is lowered from 2 mm (+ Ca2+) to 0.4 mm by spermine [8,14]. A regulatory role for these polyamines effects has not been definitively established as variation in intramitochondrial polyamine levels has not been demonstrated under specific metabolic conditions that meaningfully alter PDC activity. Elevating spermidine mimicks insulin activation of PDC in permeabilized adipocytes . PDP2 is expressed in fat synthesizing tissues  and is probably the primary target of insulin regulation, which enhances PDP activity via a mechanism that lowers the Km for Mg2+. Putative mechanisms mediating insulin regulation include allosteric mediators  and phoshorylation by PKC*.
Particularly important is the overexpression of PDK4  under conditions of starvation, which leads to a need to conserve carbohydrate. PDK4 expression is increased both by glucocorticoids and by free fatty acids via the peroxisome proliferator-activated receptor, and is blocked by an insulin-activated pathway . Impaired functioning in insulin-induced down-regulation of PDK4 (due to lack of insulin or insensitivity to insulin) deleteriously leads to overexpression of PDK4 and shutting down glucose oxidation in diabetic animals. In a complementary fashion to regulation of PDK4 expression, starvation and diabetes reduce the expression of PDP2 in rat heart and kidney (B. Huang, P. Wu, K. M. Popov & R. A. Harris, Indiana University School of Medicine, Indianapolis, IN, USA, personal communication). The expression of PDP1, the most abundant PDP in these tissues, is not affected. Re-feeding restores PDP2 expression.
To conserve carbohydrate reserves, feedback suppression of the PDC reaction when fatty acids and ketone bodies are being used as preferred energy sources results from enhanced kinase activity [1,3]. The resulting elevation of the intramitochondrial NADH/NAD+ and acetyl-CoA/CoA ratios suppresses PDC activity by effectively stimulating kinase activity, particularly PDK2 [17,18], which is expressed in most tissues . Kinase activity is reduced by the direct inhibitory effects of ADP and pyruvate and synergistically inhibited by combination of these effectors . Again, PDK2 is especially responsive to these inhibitors . Phosphate anion also enhances ADP and pyruvate inhibition of PDK2.
The focus of this review is on the functional roles of the dihydrolipoyl acetyltransferase (E2) component in eliciting the predominant changes in the operation and the effector-modulation of the PDKs (with emphasis on PDK2 and PDK3) and PDP1. In all organisms, E2 is recognized for providing the framework for assembly of the complex and integrating the sequential reactions of the complex [1,20]. In mammalian PDC, E2 also transforms kinase and phosphatase function and regulation through serving as an anchoring scaffold, an adaptor protein directly abetting efficient phosphorylation and dephosphorylation, a processing unit in translating and transmitting effector signals, and in modifying the sensitivity to alloseteric effectors that directly bind the regulatory enzymes [1,20]. Pivotal to all these roles are the dynamic interactions of the regulatory enzymes with the lipoyl domains of E2 [1,18–23]. Studies using recombinantly expressed components have been conducted over several years in our laboratory, and are supported by a large number of constructs of the E2 component, particularly involving modification of the E2 lipoyl domains.
Mammalian PDC-E2 has four globular domains (Fig. 1) with a sequential (linker region connected) set of 2 lipoyl domains at its N-terminal end. When expressed by itself, E2 assembles as a 60mer with an inner core formed through the association of 20 catalytic trimers of the C-terminal domain at the corners of a pentagonal dodecahedron. Between these C- and N-terminal domains is a small globular domain, flanked by linker regions, which binds the E1 component. In all α-keto acid dehydrogenase complexes, lipoyl domains populate the surface of the complex and consolidate the sequential five step reaction sequence by serving as substrates in the three central reactions and as mobile carriers of the intermediate forms (oxidized disulfide, 6,8-dithiol and 8-acetyl) of the lipoyl prosthetic group. The capacity of the lipoyl domains for traversing efficiently between the E1, E2 and E3 active sites is advanced by the high mobility of the extended and rather stiff Ala-Pro rich linker regions . These lipoyl domain roles and movement in support of the three complete reactions catalyzed by E1, E2 and E3 are included in Fig. 3. Apart from E2, the three domain E3-binding protein (E3BP, Fig. 1) also has an anchoring C-terminal domain, an E3-binding domain and a mobile lipoyl domain. Again, the lipoyl domain contributes as a substrate and intermediate carrier in the PDC reaction sequence and, to a lesser extent than E2 lipoyl domains, to regulatory enzyme function (below).
Activated PDK2 and PDK3 function
PDK2 exhibits the full set of the prototypical regulatory responses of mammalian PDKs described above. The E2 component transforms the efficiency of PDK2 catalysis and intervenes to create or alter all of these regulatory responses  (X. Yan, H. Bao, S. A. Kasten and T. E. Roche, unpublished results). PDK2 can phosphorylate free E1 but E2 enhances the rate of inactivation of PDC by several fold at low micromolar levels of complex and up to 5 000-fold with dilute (nm) complexes (Y. Hiromasa & T. E. Roche, unpublished results). This clearly involves PDK2 gaining efficient access to many E2-bound E1 via agile intercession of the outer domains of the E2 60mer. A combination of characterization of complexes with the analytical ultracentrifuge and kinase assays using very dilute complexes have provided important insights (Y. Hiromasa & T. E. Roche, unpublished work). PDK2 preferentially interacts with the inner lipoyl domain (L2 domain, Fig. 1) of E2 via an interaction that requires the lipoyl prosthetic group. Binding to a free L2 domain is not readily detected but reduction of the lipoyl group leads to detectable binding. Binding to two L2 in glutathione S-transferase–L2 (GST–L2) dimer structure is readily observed (Kd = 4 m) and this affinity is increased more than 10-fold upon reduction of the prosthetic group (i.e. by GST–L2red). Thus, the PDK2 dimer binds two lipoyl domains and is tightly bound by E2-E1, particularly when the lipoyl groups are reduced. Interestingly, lipoate reduction stimulates PDK2 activity (regulatory mechanism below). Even with oxidized lipoyl groups, E2 supports maximal PDK2 activity when complexes containing < 0.5 PDK2 dimers per complex are diluted to 30 nm indicating that the above affinities do not fully explain PDK2 function.
Direct binding of the free L2 domain with an oxidized lipoyl group to PDK3 is much tighter than its binding to PDK2 and has a potent effect in directly enhancing PDK3 activity . Fifty-fold lower levels of the dimeric GST–L2 domain activate PDK3 (P. Tao, Y. Hiromasa & T. E. Roche, unpublished results). A portion of the 13-fold activation of PDK3 by L2  is, in fact, due to preventing or reversing a decrease in activity of PDK3 undergoing self association in the absence of L2. Binding of two L2 or one GST–L2 dimer stabilizes PDK3 as a dimer. Short-term assays, following dilution of the L2-stabilized PDK3 dimer into assay mixtures established that excess L2 or GST–L2 still promulgates a several-fold increased kinase activity, by inducing a more active PDK3 conformation. Beyond this direct allosteric activation, the E2 60mer further enhances PDK3 activity and, in contrast to activation by L2, high activity is sustained with very dilute (< 3 nm) complexes.
The bifunctional binding to L2 is likely to underpin the ability of PDK2 and PDK3 to maintain rapid initial rates when only a few E1 bound to the E2 60mer or with dilute complexes (Y. Hiromasa & T. E. Roche, unpublished observations). To gain efficient access to E2-bound E1, successive ‘hand-over-hand’ transfer is proposed to proceed via a kinase dimer being successively bound to two lipoyl domains, randomly dissociating from one lipoyl domain and then rebinding to a second mobile lipoyl domain faster than releasing from the singly held state . Each of the lipoyl domains of E2 is concentrated within the exterior of the complex at > 1 mm[25,26], so that the rate at which a singly held dimer associates with a second lipoyl domain readily exceeds the rate for complete dissociation. This delivery mechanism may be particularly important for efficient kinase function within the mitochondrion where the high protein concentration (> 400 mg·mL−1) limits diffusion of macromolecules.
We have taken advantage of the direct activation of PDK3 by L2 to discern the surface structure of the L2 domain that engages in leveraging the conformational change in PDK3 that elicits this substantial increase in activity (X. Gong, T. Peng, and T. E. Roche, unpublished results). Modified L2 was prepared by substituting surface amino acid residues and by enzymatically inserting cofactor analogs for the lipoyl prosthetic group. As shown in Fig. 2, critical residues for PDK3 activation span > 25 Å range on L2 surface (indicated by * or **, Fig. 2). Most are located near the lipoylated end of L2 (Leu140, Asp172 and Ala174, Fig. 2A; Asp197 and Arg196, Fig. 2B). Also critically important were acidic residues (Glu162 and Glu179) located toward the other end of the domain (Fig. 2A). Even at very high levels, the well-folded and fully lipoylated Glu179→Ala–L2 did not activate PDK3, suggesting that this residue is particularly important for promoting a conformational change required for activation.
The full length of the lipoyl-lysine prosthetic group was absolutely required to in order to promulgate PDK3 activation. L2 with any of several amino acid substitutions for Lys173 failed to activate PDK3. 8-Thiol-octanoyl-L2 enhanced PDK3 activity beyond the native lipoyl-L2. Heptanoyl-Lys173-L2 inhibited PDK3 activity and effectively hindered activation by native L2. These results support the importance of interactions throughout the prosthetic group and fit the concept that extended reach of the 8-thiol group upon lipoate reduction contributes to interactions fostering kinase activation by NADH (see regulation below). The need for specific structure spread out on the surface of the L2 domain acting in concert with fully extended lipoyl lysine prosthetic group clearly implies that these extended regions work together to convert PDK3 to a more active conformation.
E2-mediated regulation of kinase activity
As indicated above, feedback suppression of PDC activity, when fatty acids and ketone bodies are primarily being consumed, results from kinase activity being greatly enhanced due to the resulting elevation of NADH/NAD+ and acetyl-CoA/CoA ratios. The rise in these ratios is sensed by the rapid and reversible E3 and E2 reactions which act to increase the proportion of the lipoyl groups of E2 and E3BP that are reduced and acetylated (Fig. 3) [22,27–29]. Short-term reduction of the lipoyl group gives rise to an 80% increase in kinase activity (PDK*), and acetylation stimulates kinase activity up to threefold (PDK**). In vitro, full stimulation can also be achieved by low levels of pyruvate reacting through the rate-limiting E1 reaction. Indeed, this approach provided important insights into the mechanism of stimulation as blocking E1 catalysis prevents reductive acetylation and consequently kinase activation. Stimulation can be observed with peptide substrates and free lipoyl domains pointing to the importance of direct allosteric interactions of the reacted lipoyl group with the kinase . 8-Thiol-octanoyl-L2 undergoes E2-catalyzed acetylation (using the inner E2-core stripped of lipoyl domains) and this acetylation stimulates kinase activity. Thus, the thiol at the 6-position of the dihydrolipoyl group is not required.
The kinases associated with purified bovine kidney complex (PDK2 and PDK3 ) were shown to respond to this control mechanism in a remarkably sensitive manner that was enhanced by ADP inhibition . Half-maximal stimulation of the activity of bovine kidney kinase is reached when < 10% of the lipoyl groups in the assembled complex are acetylated. Near-maximal stimulation of the kinase activity is attained with an NADH/NAD+ ratio of 0.1 and acetyl-CoA/CoA ratio of 0.2. The most responsive human kinase isoform is PDK2 [17,18]. The greater extended reach of the reduced and acetylated lipoyl group over that of the oxidized prosthetic group (Fig. 3) probably bestows the ability for interactions to induce activating conformations within the kinase active site. The much stronger binding of PDK2 to the L2 domain upon reduction of the lipoyl group (above) is consistent with additional interactions supporting preferential binding and eliciting a conformational change in the PDK structure.
Elevation ADP and phosphate constitute the primary metabolic indicators for a low energy state. Abundant pyruvate manifests the availability of glucose-linked substrates for solving this energy demand. Reasonably, these metabolites act together to impose a marked reduction in kinase-catalyzed inactivation of PDC activity. Elevation of K+ to physiological levels slows kinase catalysis in conjunction with decreasing the Ki for ADP. E2 activation of PDK2 activity transforms PDK2 from being poorly inhibited by pyruvate or dichloroacetate to being markedly inhibited . Inhibition results from pyruvate (or dichloroacetate) binding PDK2·ADP (and PDK2·ATP) but not the free PDK2. This results in a synergistic inhibition by pyruvate and ADP by reducing the rate of ADP dissociation (X. Yan, H. Bao, S. A. Kasten & T. E. Roche, unpublished observation). Phosphate inhibition of PDK2 is also favored and acts together with ADP and pyruvate in reducing PDK2 activity. Under conditions of physiological salts, the full set of regulatory properties of E2-activated PDK2 are consistent with an encompassing mechanism in which effectors either enhance or reduce a rate-limiting step of ADP dissociation. Accordingly, dissociation of ADP is slowed by pyruvate and speeded up upon reductive acetylation of lipoyl groups [1,18,22] (X. Yan, H. Bao, S. A. Kasten & T. E. Roche, unpublished results).
As an example of isozyme differences, pyruvate is a very weak inhibitor of PDK3. However, the combination of phosphate and ADP synergistically inhibit PDK3 activity. Human PDK3 activity is enhanced only when inhibited (e.g. by ADP) or when other conditions reduce its nonstimulated activity . For instance, PDK3 activity is increased several fold when the poorly activating L1 domain of E2 provides the only reactive lipoyl group undergoing reductive acetylation. The gain in the capacity for PDK3 activity to be enhanced by reductive acetylation in the presence of ADP and phosphate appears to be the primary basis for the higher fractional stimulation of bovine kidney kinases observed in the presence of these inhibitors .
To express human PDK4 (J. Dong, L. Hu & T. E. Roche, unpublished results), we first added a Gly-Glu-Glu amino acid sequence after the C-terminal Val-Ala-Met, as this hydrophobic sequence triggers degradation when recombinantly expressed in Escherichia coli. Subsequently we generated unmodified PDK4 in an E. coli strain lacking the ClpP proteosome. Interestingly the unmodified PDK4 differed from the GEE-modified PDK4 in being stimulated by NADH and acetyl-CoA. This suggests that the C-terminal region of PDKs is important for the lipoyl-mediated product stimulation. PDK4 is preferentially bound by the L1 domain of E2 and L3 domain of E3BP (Fig. 1) and reduction and acetylation of the lipoyl groups of these domains enhance PDK4 activity. PDK1 binds to the L1 and L2 domains of E2 (Fig. 1). PDK1 is particularly effective in phosphorylating the third phosphorylation site on E1 .
The PDK2  and branched chain dehydrogenase kinase  3D structures appear to be ideally suited for bifunctional binding of two lipoyl domains as was previously proposed based on the studies reviewed above. The PDK2 subunits have a wedge shape formed from C- and N-terminal domains. In the dimmer, the combined wedges form is located in extended apposed positions, with the extended wedge openings running in opposite directions. The monomer association involves the ATP-using C-terminal domains interacting near the base of that side of the wedge outside; the N-terminal domains form the outside of the wedges. The wedge pockets have an extended seam that extends the length of the interface between these domains with the γ-phosphate of bound ATP exposed at one end. On the reverse side from these pocket openings, the combined wedges produce a large horseshoe-shaped cavity with the outer N-terminal ends twisting in opposite directions. Significant segments of the PDK2 subunit structure were not resolved in the 3D structure, including a loop over each active site, a large C-terminal segment, and a shorter section at the N-terminus . It seems likely that lipoyl domain binding occurs either in the extended wedge pockets or within the horseshoe-shaped cavity, aided by stabilizing interactions involving one or more of the flexible termini. As noted above, the C-terminal segment may interact with the lipoyl domain or prosthetic group following reduction and acetylation of the lipoyl group because modification of the human PDK4 (by adding Gly-Glu-Glu to the C-terminus) prevented stimulation of PDK4 by NADH and acetyl-CoA but did not prevent lipoyl domain binding.
E2 mediated Ca2+-activation of PDP1
As intramitochondrial ATP decreases, free Mg2+ rises as a secondary signal of the diminished cellular energy state. In conjunction with exercise, growth, and many other neural and hormone-initiated cellular transitions that demand energy, cellular Ca2+ is elevated in the cytoplasm, leading to a concomitant rise in intramitochondrial Ca2+. PDP1 activity is considerably up-regulated in response to both of these energy-demanding up shifts in free Mg2+ and Ca2+ and facilitates an increase in the proportion of active PDC. PDP1 is a heterodimer composed of a 52-kDa catalytic subunit , PDP1c, and 96-kDa regulatory subunit . PDP1c alone has a low Km of 0.4 mm for Mg2+. However, in holo-PDP1 the regulatory subunit, PDP1r, raises the Km of the heterodimer for Mg2+ to 2 mm with Ca2+ present and 3.5 mm in the absence of Ca2+. Binding of spermine to the PDP1r subunit returns the Km of holo-PDP1 for Mg2+ to the lower level (0.5 mm). This leads to a marked increase in PDP1 activity at physiological levels of intramitochondrial free Mg2+ (0.5–1.2 mm).
E2 activation is facilitated by the Ca2+-dependent binding of PDP1, or PDP1c alone, to the L2 domain of E2 . Even at saturating Mg2+, E2 plus Ca2+ accelerates the rates of dephosphorylation by PDP1 by 10-fold and by PDP1c by sixfold. Greater-fold increases are facilitated by E2 when phosphatase activity is assessed with subsaturating levels of Mg2+ or when activities are compared with levels of free vs. E2-bound phosphorylated-E1 set at < 1 μm. We have used a set of over 30 mutant L2 domains and substitutions within the lipoyl-lysine prosthetic group to elucidate two major regions of L2 that contribute to binding PDP1 .
The first region includes the lipoyl prosthetic group and neighboring residues (Fig. 2A, ++ residues). Marked reductions in binding result from substitution of the adjacent residues Ala172 and Asp173 as well as Leu140. Full binding was retained after replacing the lipoyl group with an octanoyl group, but no activation remained with nonlipoylated L2 or following any of a series of amino acid substitutions for lipoylated Lys173. These results indicate the lipoyl cofactor probably interacts at an extended hydrophobic pocket in the surface structure of PDP1c. In contrast to the effectiveness of the octanoyl group, the dithiolane ring character of the lipoyl prosthetic group contributes to L2 binding to E1  and, as indicated above, the 8-thiol is crucial for L2 activating PDK3 . Given that the L1 domain has identical amino acids in aligned sequence positions, this region of L2 is not expected to contribute to the high specificity of PDP1 for binding to L2.
In a second region at the other end of the L2 domain, mutation of glutamates 162, 179 and 182, and glutamine 181 greatly reduces binding. Indeed, substitution of alanine or glutamine for Glu182 blocks binding of PDP1 and PDP1c to the L2 domain. As can be seen in Fig. 2A, a distinct pocket exists in the center of this set of residues. Binding of PDP1c-Ca2+ to this region may be reinforced by a resulting removal of the mutual repulsion by the three acidic residues. Only a complete 3D structure can establish how these proteins interact and elucidate whether residues such as Glu182 directly participate in forming a tight Ca2+-binding site. The conversion of the Val–Gln residues connecting Glu179 and Glu182 in L2 to the Ser–Leu sequence between equivalent acidic residues in L1 markedly reduced binding of PDP1 to L2 . This bisubstituted L2 had a substantial but lesser effect on the binding of PDP1c. The dual mutation did not reduce use of L2 in the E1 reaction despite the fact Glu179 was a key specificity residue for E1 . Five other single site mutants (Ala174→Ser and Arg196→Gln at the lipoylated end of L2, Asp213→Asn and Tyr220→Ala in the C-terminal lobe and the Tyr129→ Ala in the N-terminal segment) had greater effects on L2 binding to PDP1 than PDP1c, indicating that the PDP1r subunit of PDP1 promotes a more precise interaction with L2.
Contrary to expectations for 1 μm binding of Ca2+, a characteristic Ca2+-binding EF-hand sequence is not apparent in either PDP1c or L2. Isothermal titration calorimetry measurements revealed that Ca2+ does not bind to either L2 or PDP1c alone (A. Turkan and T. E. Roche, unpublished results). Ca2+ may play a direct bridging role in the PDP1c–L2 interaction or enhance binding by a capture mechanism. A conformational change in the PDP1c could create such a high-affinity Ca2+-binding site to stabilize the nascent interaction between the protein components. Because the highly activating binding of PDP1 to the L2 domain of E2 requires, as described above, both domain-aided hydrophobic interaction by the exterior lipoyl group at one end of L2 and electrostatic interactions at the opposite end of the L2 domain , it seems likely that these essential regions of L2 act in concert. An appealing prospect is that this extensive interaction surface of L2 supports a conformation transition that fosters and stabilizes a tight Ca2+-binding site in PDP1c. Further insight into the constitution of the Ca2+-dependent complexes formed between L2 and PDP1 or PDP1c will require detailed structures that will probably depend on crystallizing these complexes.
Among four PDK isoforms and two PDP isoforms, we have focused on PDK2, PDK3, PDP1, and its catalytic subunit, PDP1c, to illustrate how the consequential variation in the functional capacity and the responsiveness of these regulatory enzymes is dependent upon differences in their effector–modified interactions with the flexibly held outer domains of the E2 assemblage. Novel mechanisms have been uncovered whereby E2 greatly enhances kinase and phosphatase catalysis by enormously increasing access to their E1 substrates and by direct allosteric activation (particularly PDK3), mediates kinase stimulation in feedback effector control by NADH and acetyl-CoA, facilitates Ca2+-activation of PDP1, and modifies the allosteric control by effectors that bind directly to the regulatory enzymes (e.g. enhances pyruvate inhibition PDK2 and alters the Mg2+ requirement of PDP1). Requisite interactions of PDK3 and PDP1 with L2, the inner lipoyl domain of E2, are shown to engage extensive regions of the surface of L2 and to require the lipoyl prosthetic group and, in the case of the PDKs, to be markedly modified by reaction of this prosthetic group.
This work was supported by the National Institutes of Health Grant DK18320 and by the Kansas Agriculture Experiment Station – contribution 03-27-J.