Báo cáo khoa học: Essential roles of lipoyl domains in the activated function and control of pyruvate dehydrogenase kinases and phosphatase isoform 1 pot

Kasten, Haiying Bao and Jianchun Dong Department of Biochemistry, Kansas State University, Manhattan, Kansas, USA Four pyruvate dehydrogenase kinase and two pyruvate dehydrogenase phosph

M I N I R E V I E W

Essential roles of lipoyl domains in the activated function

and control of pyruvate dehydrogenase kinases

and phosphatase isoform 1

Thomas E Roche, Yasuaki Hiromasa, Ali Turkan, Xiaoming Gong, Tao Peng, Xiaohua Yan,

Shane A Kasten, Haiying Bao and Jianchun Dong

Department of Biochemistry, Kansas State University, Manhattan, Kansas, USA

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

(non-phosphorylated) pyruvate dehydrogenase component

Necessary adaptations of PDC activity with varying

meta-bolic 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

Keywords: pyruvate dehydrogenase complex; PD kinase;

PD phosphatase; dihydrolipoyl acetyltransferase; lipoyl domain

Introduction

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-tissue-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 pro-portion 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 [8] 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 [1], 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

Correspondence to 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: bchter@ksu.edu

Abbreviations: PDC, pyruvate dehydrogenase complex; E1, pyruvate

dehydrogenase component; E2, dihydrolipoyl acetyltransferase

component; L1 domain, NH 2 -lipoyl domain of E2; L2 domain,

interior lipoyl domain of E2; PDK, pyruvate dehydrogenase kinase;

PDP, pyruvate dehydrogenase phosphatase; PDP1c, catalytic subunit

of PDP1; PDP1r, regulatory subunit of PDP1; E3, dihydrolipoyl

dehydrogenase; E3 BP, E3-binding protein; L3, N-terminal lipoyl

domain of E3 BP; GST, glutathione S-transferase.

(Received 16 July 2002, revised 27 December 2002,

accepted 20 January 2003)

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

Kmvalues for Mg2+of both PDP isoforms The Kmof PDP2

in the absence of polyamines is very high (16 mM) and

reduced to 3 mM[7]; whereas the Kmof PDP1 for Mg2+is

lowered from 2 mM( + Ca2+) to 0.4 mMby 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 [4] PDP2 is expressed in fat

synthesizing tissues [7] and is probably the primary target

of insulin regulation, which enhances PDP activity via a

mechanism that lowers the Kmfor Mg2+ Putative

mecha-nisms mediating insulin regulation include allosteric

media-tors [15] and phoshorylation by PKC* [16]

Particularly important is the overexpression of PDK4 [2]

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

activated pathway [2] 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

stimula-ting kinase activity, particularly PDK2 [17,18], which is

expressed in most tissues [17] Kinase activity is reduced by

the direct inhibitory effects of ADP and pyruvate and

synergistically inhibited by combination of these effectors

[19] Again, PDK2 is especially responsive to these

inhi-bitors [18] 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

phospha-tase function and regulation through serving as an

anchor-ing scaffold, an adaptor protein directly abettanchor-ing 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 recom-binantly 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 a-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 [24] 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)

Fig 1 E2 and E3BP domains and their binding interactions E2 subunit domains: L1, N-terminal lipoyl domain; L2, inner lipoyl domain; B, E1 binding domain; I, oligomer-forming, acetyl-transferase-catalyzing inner domain E3BP domains: L3, N-terminal lipoyl domain; B, E3 binding domain; I, inner domain which associates with the inner domain of E2 Dotted connections indicate binding interactions to the catalytic components (E1 and E3), kinase isoforms (PDK1, PDK2, PDK3, PDK4) and Ca 2+ -binding of PDP1.

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

[18] (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

ultracen-trifuge 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

struc-ture is readily observed (Kd¼ 4M) and this affinity is

increased more than 10-fold upon reduction of the

pros-thetic 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

Interest-ingly, lipoate reduction stimulates PDK2 activity

(regulat-ory 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 [18] 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 [18] 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 [21] 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 mgÆmL)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 A˚ 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 Glu179fiAla–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 effect-ively 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 stimu-lates 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 [22]

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 [18]) were shown to respond

to this control mechanism in a remarkably sensitive manner that was enhanced by ADP inhibition [28] 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

Fig 2 Surface residues of the L2 domain of E2 that are required for activating PDK3 and PDP1 Panels A and B show opposing sides of space-filled models of the human L2 domain The position of Lys173 (lipoylated lysine) is at the top of the structures; unstructured regions that are not part of the connecting linker regions are shown at the N- and C-termini at the bottom of the structures Lys173 must be lipoylated for an L2 construct to bind and activate PDK3 or bind and competitively prevent E2 activation of PDP1 ** or * designate resi-dues whose substitution removes 80% or 50%, respectively, of L2 activation of PDK3 (X Gong, T Peng, and T E Roche, unpublished results) In these panels, ++ or + indicates a mutation that reduces PDP1 binding to L2 by 75% or 45%, respectively [32].

Fig 3 Signal mechanism for stimulation of PDKactivity by elevation of the NADH/NAD+and acetyl-CoA/CoA ratios In the PDC reaction (forward direction proceeds in the counterclockwise direction), the rapid and reversible E3 and E2 reactions respond to changes in these products-to-substrate ratios and react to thereby determine the pro-portion of lipoyl groups that are in the oxidized, reduced or acetylated forms When a kinase dimer binds to a lipoyl domain with its lipoyl group in the oxidized form (bound PDK, top left), E2 facilitates higher rates of phosphorylation of the E2-bound E1 Binding of a kinase dimer to a lipoyl domain containing a reduced lipoyl group fosters a further increase in kinase activity (PDK* state) Binding of a PDK dimer to a lipoyl domain with its lipoyl group reductively acetylated yet additionally enhances kinase activity (PDK** state) See the text for the magnitude of these stimulatory effects on PDK activity.

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

sub-strates 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 Kifor ADP E2 activation

of PDK2 activity transforms PDK2 from being poorly

inhibited by pyruvate or dichloroacetate to being markedly

inhibited [18] Inhibition results from pyruvate (or

dichlo-roacetate) binding PDK2ÆADP (and PDK2ÆATP) but not

the free PDK2 This results in a synergistic inhibition by

pyruvate and ADP by reducing the rate of ADP

dissoci-ation (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 [18] For instance, PDK3 activity is increased several

fold when the poorly activating L1 domain of E2 provides

the only reactive lipoyl group undergoing reductive

acety-lation 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 [18]

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

recombi-nantly 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

stimula-ted 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 [30]

The PDK2 [11] and branched chain dehydrogenase

kinase [12] 3D structures appear to be ideally suited for

bifunctional binding of two lipoyl domains as was

previ-ously 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 direc-tions 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 c-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 [11] 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 inter-act 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+[4] 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 [13], PDP1c, and 96-kDa regulatory subunit [31] PDP1c alone has a low Km of 0.4 mM for Mg2+ [13] However, in holo-PDP1 the regulatory subunit, PDP1r, raises the Kmof the heterodimer for Mg2+to 2 mMwith

Ca2+ present and 3.5 mM in the absence of Ca2+ [14] Binding of spermine to the PDP1r subunit returns the Kmof 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 [23] 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 lM 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 [32]

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 [33] and, as indicated above,

the 8-thiol is crucial for L2 activating PDK3 [23] 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 [32] 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 [33] Five other single site mutants (Ala174fiSer and

Arg196fiGln at the lipoylated end of L2, Asp213fiAsn

and Tyr220fiAla in the C-terminal lobe and the Tyr129fi

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 lM 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 [32], 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

Conclusion

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 regula-tory 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+ -activa-tion 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

Acknowledgements

This work was supported by the National Institutes of Health Grant DK18320 and by the Kansas Agriculture Experiment Station – contribution 03-27-J.

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