(Received for publication, April 10, 1995; and in revised form, August 18, 1995)
From the
To conserve carbohydrate reserves, the reaction of the pyruvate
dehydrogenase complex (PDC) must be down-regulated when the citric acid
cycle is provided sufficient acetyl-CoA. PDC activity is reduced
primarily through increased phosphorylation of its pyruvate
dehydrogenase (E1) component due to E1 kinase
activity being markedly enhanced by elevated intramitochondrial
NADH:NAD and acetyl-CoA:CoA ratios. A mechanism is
evaluated in which enhanced kinase activity is facilitated by the
build-up of the reduced and acetylated forms of the lipoyl moieties of
the dihydrolipoyl acetyltransferase (E2) component through
using NADH and acetyl-CoA in the reverse of the downstream reactions of
the complex. Using a peptide substrate, kinase activity was stimulated
by these products, ruling out the possibility kinase activity is
increased due to changes in the reaction state of its substrate, E1 (thiamin pyrophosphate). Each E2 subunit contains
two lipoyl domains, an NH
-terminal (L1) and the inward
lipoyl domain (L2), which were individually produced in fully
lipoylated forms by recombinant techniques. Although reduction and
acetylation of the L1 domain or free lipoamide increased kinase
activity, those modifications of the lipoate of the kinase-binding L2
domain gave much greater enhancements of kinase activity. The large
stimulation of the kinase generated by acetyl-CoA only occurred upon
addition of the transacetylase-catalyzing (lipoyl domain-free) inner
core portion of E2 plus a reduced lipoate source, affirming
that acetylation of this prosthetic group is an essential mechanistic
step for acetyl-CoA enhancing kinase activity. Similarly, the lesser
stimulation of kinase activity by just NADH required a lipoate source,
supporting the need for lipoate reduction by E3 catalysis.
Complete enzymatic delipoylation of PDC, the E2-kinase
subcomplex, or recombinant L2 abolished the stimulatory effects of NADH
and acetyl-CoA. Retention of a small portion of PDC lipoates lowered
kinase activity but allowed stimulation of this residual kinase
activity by these products. Reintroduction of lipoyl moieties, using
lipoyl protein ligase, restored the capacity of the E2 core to
support high kinase activity along with stimulation of that activity up
to 3-fold by NADH and acetyl-CoA. As suggested by those results, the
enhancement of kinase activity is very responsive to reductive
acetylation with a half-maximal stimulation achieved with 20% of
free L2 acetylated and, from an analysis of previous results, with
acetylation of only 3-6 of the 60 L2 domains in intact PDC. Based
on these findings, we suggest that kinase stimulation results from
modification of the lipoate of an L2 domain that becomes specifically
engaged in binding the kinase. In conclusion, kinase activity is
attenuated through a substantial range in response to modest changes in
the proportion of oxidized, reduced, and acetylated lipoyl moieties of
the L2 domain of E2 produced by fluctuations in the
NADH:NAD
and acetyl-CoA:CoA ratios as translated by
the rapid and reversible E3 and E2 reactions.
In mammalian cells, the pyruvate dehydrogenase complex (PDC) ()controls the oxidative utilization of glucose(1) .
Flux through this reaction results in a net depletion of body
carbohydrate reserves. The activity of PDC must be reduced when fatty
acids or ketone bodies are being preferentially used to provide
2-carbon units for oxidative energy production by citric acid
cycle/oxidative phosphorylation systems, a routine situation in many
organs. Furthermore, under conditions of starvation or diabetes, the
activity of PDC is reduced to a minimal level to conserve carbohydrates
essential for the brain and other specialized tissues/organs. To
achieve its critical role in cellular fuel conservation, the PDC
reaction is controlled primarily by a highly regulated
phosphorylation/dephosphorylation cycle which is carried out by
dedicated kinase and phosphatase components. Phosphorylation of the
pyruvate dehydrogenase (E1) component inactivates the complex
and dephosphorylation reactivates the complex.
Shortly after PDC was
shown to be regulated by this interconversion (2) , the
capacity of fatty acids and ketone bodies to promote inactivation of
PDC was demonstrated in intact tissues (3, 4) and in
studies with intact mitochondria(5, 6) . Studies with
purified complex (7, 8, 9) and isolated
mitochondria (10, 11, 12) found that
increases of the PDC reaction products promote an increase in the
proportion of PDC in the phosphorylated (inactive) state. With purified
complex, the activity of the kinase is greatly enhanced upon elevation
of the NADH:NAD ratio and the acetyl-CoA:CoA ratio
along with a reciprocal reduction in phosphatase activity as the
NADH:NAD
ratio is elevated(7) .
Accordingly, the E1a kinase has a crucial role wherein it throttles down PDC activity in response to increases in the mitochondrial acetylation and reduction potentials. Not only are NADH and acetyl-CoA produced during mitochondrial oxidation of all fuels, they are direct products of the PDC reaction. This laboratory presented evidence that this control of kinase activity initially involves these product to substrate ratios being translated by competitive utilization in the downstream reactions of the complex which, in turn, adjusts the fraction of the complex's lipoyl prosthetic groups in the oxidized versus reduced versus acetylated forms(13, 14, 15, 16, 17) . Specifically, our mechanism proposes that kinase down-regulates PDC activity due to NADH reacting in the reverse of the dihydrolipoyl dehydrogenase (E3) reaction and acetyl-CoA reacting in the reverse of the dihydrolipoyl acetyltransferase (E2) reaction. Typically a 60-80% enhancement in kinase activity occurs upon lipoate reduction and up to a 3-fold enhancement following lipoate acetylation. The potential for understanding the molecular basis of this control has greatly improved with new insights into the structure of E2 subunits and the unusual nature of the association of the kinase with E2.
The E2 subunits of mammalian
PDC have four domains connected by relatively large (2-3 kDa) and
highly mobile linker regions Fig. 1(18, 19, 20) . Sixty
COOH-terminal inner domains (E2) associate to form
a dodecahedral inner core which catalyzes the transacetylation
reaction. Each inner domain is connected to 3 globular domains by
linker or hinge regions. The globular domains consist of two
10-kDa lipoyl domains (E2
and E2
, or L1 and L2) and an E1-binding
domain (E2
) located between the inner core and the
lipoyl domain region.
Figure 1:
Domain structure of the dihydrolipoyl
acetyltransferase (E2) component and interactions with the
kinase and E1 components. E2 has four globular
domains consisting of an NH-terminal lipoyl domain, L1, an
inner lipoyl domain, L2, an E1 binding domain, and a
core-forming, transacetylase-catalyzing inner domain at the
COOH-terminal end(18, 19, 20) . The kinase
binds to the L2 domain by a domain-specific and lipoyl prosthetic
group-requiring interaction(26) . E1, an
tetramer, binds to the B domain of E2 via its
-subunit (18) .
The subunits of E1a kinases of PDC
and the branched-chain -keto acid dehydrogenase complex are
related to procaryotic histidine kinases but not to the
extramitochondrial serine and tyrosine kinases of
eukaryotes(21, 22, 23) . PDC kinase binds to
the lipoyl domain region of E2 through an association that
requires the lipoyl prosthetic
group(24, 25, 26) . Using lipoylated and
delipoylated forms of recombinant L1 and L2 of human
PDC-E2(27) , the kinase was shown to bind
preferentially to the lipoylated L2 (Fig. 1)(26) . The E1a kinase was also shown to interchange rapidly between L2
structures(26) . To account for tight binding and rapid
interchange, a dynamic ``hand over hand'' mechanism is
proposed in which a dimeric kinase alternates between being bound to
one and two L2 domains. This interchange and catalytic function of the
kinase exert their combined effects in the limited space at the surface
of the complex where the a kinase molecule and many E1
components are tightly bound to the mobile outer domains of E2. The capacity of continuously bound kinase to phosphorylate
bound E1 components more rapidly than free kinase can
phosphorylate free E1 is termed E2-activated kinase
function.
The aim of this work is to determine the compulsory
components, domains, and catalytic processes involved in kinase
stimulation by NADH and acetyl-CoA, and to evaluate the relative
capacities of the recombinant L1 and L2 in mediating the stimulation of
kinase in the presence or absence of E2-activated kinase
function. We have found that stimulation occurs with a peptide
substrate of the kinase; that a lipoyl source must be available for
catalytic reduction by E3 or acetylation by E2; that the L2 lipoyl domain is much more
effective in mediating kinase stimulation than the L1 domain; and that
the kinase is remarkably sensitive to the level of acetylation of L2.
The ``Discussion'' integrates these observations and draws
new mechanistic conclusions based on these and previous results.
The fractional binding of TPP to E1 can be evaluated in PDC activity assays because bound TPP
is continuously converted to the more tightly bound
hydroxyethylidene-thiamin pyrophosphate intermediate, the substrate of
the rate-limiting reductive acetylation step(35, 36) .
The levels of TPP bound to E1 were evaluated by comparing the
rates of PDC reaction obtained by diluting enzyme 200-fold as the last
addition into assay mixtures lacking or containing TPP. Reconstituted
PDC activity was measured with resolved E1 and excess E2X
K1K2 subcomplex (10 µg) and E3 (4
µg) and limiting E1 (4 µg). The fractional E1-TPP is defined as the ratio of the highest activity
observed in the absence of TPP to that observed in the presence of TPP.
The effects of changes in NADH:NAD and
acetyl-CoA:CoA ratios on PDC activity were analyzed in the absence of
ATP in the same high K
buffer (buffer A) used to
evaluate the regulation of kinase activities rather than the standard
40 mM potassium phosphate buffer used in routine PDC activity
assays. Pyruvate and TPP were introduced at saturating levels of 1.0
mM and 0.2 mM, respectively. When the relative amount
NADH + NAD
were varied while retaining a total
concentration of constant 0.5 mM, PDC activity was measured at
365 nm (
= 4.2
OD
mM
cm
rather than at 340 nm. Other conditions were as described in the legend
to Fig. 8.
Figure 8:
Effects of changes in product to substrate
ratios on PDC reaction measured in buffer A. All reactions contained
2.0 mM pyruvate and 0.1 mM TPP. Reaction mixtures
were modified as follows. The acetyl-CoA:CoA ratio was varied at a
constant total pool of 0.2 mM in presence of 1.0 mM NAD (
) or at a 0.1 NADH:NAD
ratio using 1.1 mM total pyridine nucleotides (
);
the NADH:NAD
ratio was varied with a constant total
pool of 0.5 mM with 0.2 mM CoA (
) or at an
acetyl-CoA:CoA ratio of 1 (
) using 0.1 mM of each. In
each case 100% rates were the rate observed at a product to substrate
ratio of zero for the varied ratio. Other conditions and assay
procedures are described under ``Experimental
Procedures.''
Table 1also shows that E1 can be
replaced by peptide substrate so the stimulations are not dependent on
changes in E1(38) . ()This is consistent
with the observation of Reed et al.(40) that kinase
stimulation occurs with tryptic digestion of PDC. Such PDC digestion
produces a complex mixture of lipoyl domains, the catalytic inner core
of E2 and the trypsin-resistant E3, all of which are
required for kinase stimulation (see below).
Because some
stimulation by acetyl-CoA is detected in the absence of NADH, the level
of incorporation of covalently attached acetyl groups from
[1-C]acetyl-CoA was determined in the presence
or absence of unlabeled ATP under the conditions of kinase assays (Table 1). Without NADH, exposure to submillimolar levels of
dithiothreitol allows acetylation of a small portion of the lipoyl
groups by acetyl-CoA. The low stimulation of kinase that occurs in
conjunction with the low level of acetylation in the absence of NADH
agrees with previous studies (15, 16, 17) (cf.
``Discussion''). Thus, our results eliminate the possibility
that kinase stimulation is due to changes in the form of TPP
interacting with the E1 substrate but they are consistent with
the participation of lipoyl prosthetic groups.
Figure 2:
Effect of lipoamidase treatment on the
activity and effector stimulation of E1a kinase associated
with intact PDC. Lipoamidase treatments of PDC were for 150 min for
second series of bar graphs (PDC-D
) and
for 210 min in the fourth set of bar graphs (PDC
-D
).
The interested preparations (PDC
and PDC
) were incubated for the same times
with lipoamidase. No changes in the SDS-PAGE profiles were detected
with any of the incubated samples. Kinase assays were conducted as
described under ``Experimental Procedures'' with the
addition, as indicated by the bar graph key legend, of NADH and
NAD
in a mixture to a final concentration of 0.6 and
0.2 mM, respectively, and of acetyl-CoA to 50 µM.
Pyridine nucleotides were added 60 s and acetyl-CoA 20 s prior to
[
-
P]ATP, and the reactions were terminated
after 60 s.
Using a 210-min lipoamidase treatment of
PDC lowered the level of acetylation 1.15 ± 0.3 in
PDCD
from 162.8 (
)for the
untreated PDC
control, and abolished the stimulations of
kinase activity by NADH and/or acetyl-CoA relative to the control (Fig. 2). The low stimulation of control kinase of
PDC
or PDC
by acetyl-CoA in the absence of NADH
was associated with a low level of acetylation of lipoates reduced
during storage of the complex in 0.5 mM dithiothreitol.
SDS-PAGE analysis of the lipoamidase treated PDC
or
PDC
showed no changes in the protein pattern indicating
that there was no proteolysis during the lipoamidase treatments.
Figure 3:
Effects on kinase activity and regulation
of enzymatic removal and restoration of lipoyl groups of the E2X
kinase subcomplex. The E2
X
kinase subcomplex was treated with lipoamidase
for 210 min, and the lipoamidase was inactivated with PMSF as described
under ``Experimental Procedures.'' Lipoyl groups were then
restored to a portion of this preparation using E. coli lipoyl
protein ligase and then substrates removed as described under
``Experimental Procedures.'' The control sample of subcomplex
was given parallel incubations. Kinase assays included 25 µg of E1; pyridine nucleotides were added 60 s and acetyl-CoA 20 s
prior to [
-
P]ATP, and the reactions were
terminated after 60 s.
Figure 4: Capacity of the L1 and L2 domains of E2 to support effector stimulation of kinase activity. Kinase assays were conducted with 25 µg of E1 containing low kinase, and, as indicated, human recombinant L1 or L2 domains were included at 16 µM. Effectors were added at the levels and times given in Fig. 3. The solid bar shows kinase activity in the absence of L1 or L2. Other conditions were as described under ``Experimental Procedures.''
In parallel experiments, the levels of
acetylation of lipoyl domains under the conditions used for studying
kinase stimulation were evaluated. With the L2 domain at 20 s (time of
ATP addition after acetyl-CoA addition) and at 80 s (the time the
kinase reactions were terminated) 0.11 and 0.25 acetyl groups were
incorporated per L2 in the presence of E2 but in
the absence of NADH. Whereas in the presence of
NADH/NAD
, slightly more than one acetyl group per L2
was incorporated at 20 and 80 s. Similar levels of acetylation occurred
with L1. This indicates that there is a low level of biacetylation (i.e. at the 6- and 8-positions of a lipoate) as previously
observed with the recombinant L1 and L2 constructs(27) .
Figure 5:
Relationship between the degree of
acetylation of the L2 domain and stimulation of kinase activity. In
kinase and acetylation assays, the acetylation of 16 µM L2
catalyzed by the E2 oligomer was carried out with
removal of CoA formed by the
-ketoglutarate dehydrogenase complex
reaction under the conditions described under ``Experimental
Procedures.'' Acetyl-CoA was included at levels from 1.5 to 30
µM and was completely consumed in acetylating L2 at all
but the two highest concentrations (based on the measured extents of
acetylation). NADH and NAD
were present at 0.25
mM. Other conditions were as described under
``Experimental Procedures.''
Figure 6: Relative effects of dihydrolipoamide and acetylated-dihydrolipoamide to L1 and L2 on kinase activity and capacity of dihydrolipoamide to support lipoyl domain effects. Where indicated by key bar legends, dihydrolipoamide, L1, and L2 were added to assays at 20 µM. E1-kinase was added to reaction mixtures containing 1 µg of E3 and all reactants except dihydrolipoamide, acetyl-CoA, and ATP. Dihydrolipoamide was added 40 s and acetyl-CoA 20 s before ATP, and other additions were made as described in the legend to Fig. 4and under ``Experimental Procedures.''
Dihydrolipoamide
alone was effective in supporting acetyl-CoA stimulation. It was
important that acetylation of dihydrolipoamide immediately precede the
kinase assay since stimulation was not detected with stored
preparations in which acetyl groups transfer from L-isomer to
incorrect D-isomer and from 8-position to 6-position of the
dihydrolipoamide. Dihydrolipoamide replaced NADH in supporting the
higher acetyl-CoA stimulation with L1 and particularly with L2 (diagonally cross-hatched bars). Acetyl-CoA stimulation again
required E2 (minus E2
results
not shown). For the marked stimulation by acetyl-CoA plus L2,
dihydrolipoamide likely reduced enough L2 lipoates in the presence of E3 to allow L2-acetylation to an extent giving near maximal
stimulation. Indeed, the 4.7-fold enhancement was greater than with
NADH probably due to the extent of acetylation being somewhat lower,
resulting in less diacetyl-L2. Although reduction and acetylation of
the lipoate of L2 is clearly most effective, a direct stimulation of
kinase activity is observable with dihydrolipoamide and
acetyl-dihydrolipoamide and this limits mechanistic possibilities (cf. ``Discussion'').
Figure 7: Effects of removal and restoration of the lipoyl groups of L2 on kinase stimulation by NADH and acetyl-CoA. The L2 domain was delipoylated with lipoamidase and relipoylated with lipoyl protein ligase using the conditions described under ``Experimental Procedures.'' Kinase assays were conducted as described in the legend to Fig. 4and under ``Experimental Procedures,'' except that L2 was unchanged, delipoylated, or relipoylated, as indicated.
Randle and co-workers (45, 46) pointed out
the importance of feedback control of PDC in satisfying metabolic needs
and presented evidence for direct product inhibition of the PDC
reaction. After regulatory interconversion of PDC between active and
inactive forms (2) became known, Randle's laboratory
contributed to evidence (cf. Introduction) that the products
of PDC reaction influence the proportion of PDC in the active form,
through diverse studies with purified complex isolated mitochondria,
and intact tissues (1, 8, 9, 47) .
In their studies with purified porcine heart PDC, no stimulation by
acetyl-CoA beyond that of NADH was detected, and they speculated that
enhanced kinase activity might be due to the removal of an inhibition
of kinase activity caused by the absence of an interaction between
oxidized lipoate and the E1 substrate (8, 9) . Although not the correct mechanism and
detection of an effect of acetyl-CoA required use of higher
K concentration (see below), the suggestion of a role
for changes in the intermediate status of lipoyl groups was insightful
as was their linking of the stimulatory effect of pyruvate (41) to the effect of products(9) . This laboratory
presented the initial and much subsequent evidence that reduction and
acetylation of lipoyl prosthetic groups of PDC constituted essential
steps in the operation of a sensitive signal translation process
whereby increases in the NADH:NAD
and acetyl-CoA:CoA
ratios markedly enhance kinase
activity(13, 14, 15, 16, 17) .
However, elucidation of the minimal requirements for the operation of
this control could be accomplished only following a greatly enhanced
understanding of the organization of the complex, the preparation of
individual lipoyl domains of PDC-E2, and the availability of
lipoamidase and lipoyl protein ligase, combined with the selective use
of peptide substrate and free forms of lipoamide.
Using these tools, we have found that the marked enhancement of kinase activity by acetyl-CoA requires a lipoate source and its reduction, the catalytic domain of E2, and a peptide substrate. These requirements conclusively support a change in the kinase mediated by catalytically forming acetyl-dihydrolipoate. Accumulation of this intermediate explains the similarly strong stimulation of kinase activity by low pyruvate via E1(TPP) catalysis. The direct stimulation by dihydrolipoamide, its capacity to replace NADH in potentiating acetyl-CoA stimulation, and the complete lack of an effect of NADH on kinase activity in the absence of a lipoate source strongly favor NADH boosting kinase activity through its use in the E3 reaction. We have established that this intervention is abolished by complete removal of lipoates from intact E2 subunits or lipoyl domains. Of particular importance for understanding the operation and regulation of the kinase is our finding that the kinase-binding L2 domain is much more effective than E2's L1 domain or lipoamide in mediating kinase stimulation. The 3-fold increase in kinase activity generated by reductive acetylation of recombinant L2 is comparable to the change in kinase activity produced by acetylation of lipoyl moieties in the intact complex. Reduced L1 was only slightly more effective than free dihydrolipoamide when compared at 20 µM, and 50 µM dihydrolipoamide gave at least an equivalent stimulation, suggesting the structure of L1 does not contribute significantly to these effects. At higher levels L1 inhibits kinase activity(26) . Thus, the L2 domain must have structural features that facilitate its dedicated roles in binding the kinase, producing enhanced E1 phosphorylation within the confines of the complex, and further increasing kinase activity upon reduction and acetylation of its lipoyl group. This raises the question of the linkage between these L2-supported actions of the kinase.
Detaching
lipoates of the E2 core removes the capacity of
the E2 core to give a severalfold increase in E1
phosphorylation(24) . Via lipoyl-dependent binding to the L2
domain, rapid ``hand over hand'' interchange of a kinase
dimer between lipoyl domains apparently eliminates constraints normally
associated with binding that is as tight as exists between E2
and the kinase(25, 26) . It is significant that a
lipoamidase treatment of PDC, which left only a few lipoates and caused
close to full loss of this E2-enhanced kinase function, still
allowed marked stimulation of the residual kinase activity upon
acetylation of those few domains retaining lipoyl groups (PDC
series in Fig. 2). This result forcefully suggests that
the kinase has moved to the limited number of lipoylated L2 domains
and, furthermore, that their acetylation (during a short-lived
dissociation and reassociation of the kinase) facilitated kinase
stimulation. Based on this continued stimulation when very few E2 subunits retain lipoate, we hypothesize that maximal
effector stimulation is mediated by an allosteric effect induced by a
reductively acetylated L2 domain that becomes engaged in binding of the
kinase (i.e. not by interaction of an acetylated prosthetic
group on a neighboring lipoyl domain that is not engaged in binding the
kinase). Such a highly specific interaction is further supported below.
Beside E1 catalyzed reductive-acetylation giving a nearly
equivalent stimulation to
acetyl-CoA(13, 14, 15) , -ketobutyrate (38) gives a lesser stimulation similar to that of
propionyl-CoA at low levels of propionylation(16) . In the
absence of a lipoyl domain source, kinase phosphorylation of E1 is not stimulated at low pyruvate ((17) , miniprint
section); and here we show that TPP and a lipoyl domain source are
needed to achieve pyruvate stimulation. These findings raise the
question as to whether the E1 reaction or the downstream (E2,E3) reactions determine the relative proportion
of lipoates in the oxidized, reduced and acetylated forms in the
complex. Since E1 catalyzes the rate-limiting
step(35, 36) , under most metabolic conditions, this
distribution should be determined by the NADH:NAD
and
the acetyl-CoA:CoA ratios and kinase activity should primarily respond
to their fluctuation.
With purified porcine liver and bovine kidney
PDC, half-maximal stimulations of kinase activities occur at ratios of
NADH:NAD of 0.03 and 0.05, respectively, and, with 2
mM dithiothreitol as a reducing agent, at ratios of
acetyl-CoA:CoA of 0.1 and 0.17, respectively(28) . Even lower
acetyl-CoA:CoA (<0.1) give half-maximal stimulation when a
NADH:NAD
ratio of 0.1 is provided to reduce lipoates
(data not shown). At these low ratios, there is minimal product
inhibition of the PDC reaction by NADH plus acetyl-CoA (cf.
the 0.1 value on the
curve, Fig. 8). Significant direct
product inhibition occurs with low NADH:NAD
ratio when
the acetyl-CoA:CoA ratio was held at a relatively high level of 1 (Fig. 8,
). Acetyl-CoA:CoA ratios have been generated at
and above this range with isolated mitochondria and are proposed to
contribute to direct PDC inhibition (48) ; depletion of CoA may
also inhibit PDC due to buildup of fatty acyl-CoAs(49) . While
these probably contribute to a fine tuning role on a small portion of
active PDC, enhanced phosphorylation should effectively throttle down
PDC activity before onset of these extremes.
A priori, the
lesser enhancement of kinase activity by just the conversion of
lipoates from the oxidized to the reduced form could involve either
removal of an inhibitory interaction of oxidized lipoates, an
enhancement of kinase activity by gain of a positive allosteric
interaction of the reduced form, or a change in the thiol disulfide
state of a kinase subunit. Studies with E2-bound kinase found
that the kinase is sensitive to thiol
reagents(13, 52) , effects probably due to changes in
the oxidation-reduction state of the lipoate of L2 domains. Although
there is a small increase in alkylation of cysteines of the K1 subunit
following treatment of E2X
kinase-E3 with
NADH, this was not rapidly reversed by excess NAD
which opposes kinase stimulation. (
)Thus, a mediator
role for changes in the thiol-disulfide status of the kinase is not
indicated. Furthermore, our finding that free dihydrolipoamide and to a
greater extent acetylated dihydrolipoamide directly stimulated kinase
activity in the absence of a lipoyl domain source, eliminating the
prospect that stimulation results from removal of an inhibitory effect
by the oxidized form of lipoate. As modeled in Fig. 9, we
conclude that kinase activity is enhanced through direct positive
allosteric interactions of the reduced or the acetylated form of an L2
lipoate and we further suggest that L2 specificity derives from the L2
domain engaged in binding the kinase.
Figure 9:
Model of proposed steps in kinase
stimulation. The proportion of L2 domains of the E core having oxidized, reduced, or acetylated lipoates responds to
changes in the NADH:NAD
ratio and acetyl-CoA:CoA ratio
via the rapid and reversible reactions catalyzed by E3 and E2, respectively. Our model proposes that a change from the
kinase binding to one or two L2 containing only oxidized lipoate
(nonstimulated K state) to interacting with an L2 containing a
reduced or an acetylated lipoate results in the modified lipoates
allosterically inducing conformational changes that generate the
progressively more active K* or K** states,
respectively.
Consistent with stimulation
occurring at low ratios of acetyl-CoA to CoA, half-maximal and
near-maximal stimulation of kinase associated with the intact bovine
kidney PDC are achieved with only 7-10 and 22-26 acetyl
groups incorporated, respectively, per
PDC(15, 16, 17) . With the recent knowledge
that there are 3 lipoyl domains, two on E2 and one on the E3-binding protein giving a total of at least 126 lipoates per E2, ()it seems likely that no more than 12 and
possibly as few as 7 acetyl groups are incorporated per PDC into the
kinase-binding L2 domain for near-maximal stimulation, and as few as 3
and no more than 6 are incorporated for half-maximal stimulation. (
)This constitutes a highly responsive regulatory mechanism
in which the kinase must preferentially interact with acetylated L2 and
suggests that full stimulation may result from only one subunit of a
kinase dimer interacting with a reductively acetylated L2 domain.
Consistent with this prospect, half-maximal stimulation occurred with
free L2 when only 20% of the L2 are acetylated. Although a much higher
portion was found than estimated (above) for the intact complex, very
different conditions are operative at the outer surface of the complex
where lipoyl domains are concentrated at
1 mM, making the
localized L2 level at least 50-fold higher than the highest level of
free L2 used in the present work. Interaction of the kinase with
acetylated-L2 would be aided by steady state turnover in the overall
PDC reaction rapidly changing the location of acetyl groups within the
multienzyme cluster. Kinase shuffling between L2 domains may also
contribute, but this is needed, regardless, since acetylation by
acetyl-CoA requires an unhindered lipoate(50, 51) ,
and the kinase must interact after L2 acetylation. We conclude that
increased reductive-acetylation is very effective in activating kinase
due to this preferred interaction of acetylated L2 altering the
structure of the kinase and thereby increasing kinase activity (Fig. 9).
In being stimulated, kinase kinetic properties must
fundamentally change since stimulation occurs over a wide range of
rates (e.g. even with a peptide substrate that is slowly
phosphorylated) and in the absence of kinase movement on the surface of
the E2 core. Previous studies demonstrated that stimulation of
the kinase requires physiological levels of potassium
salts(7, 14, 53) . Increasing K from low (0-10 mM) to higher levels (90-120
mM) leads to a marked inhibition of kinase activity and a gain
in the capacity for NADH and acetyl-CoA stimulation. Another property
of the kinase that changes with increasing K
ion is
that ADP inhibition markedly increases(55) . A potential
mechanism is that interaction of the kinase with an L2 domain
containing an acetyl-dihydrolipoate leads to a conformational change in
the kinase that speeds up ADP dissociation, which is probably the
rate-limiting step in the kinase reaction at elevated (physiological)
K
levels. Consistent with that prospect are the
observation that acetylation causes a greater stimulation of
ADP-inhibited kinase (13) and the evidence that kinase-ADP is a
prominent reaction intermediate, since pyruvate effectively inhibits by
binding to the kinase-ADP in the absence of free ADP(56) . For
this mechanism to be correct, it must operate in conjunction with the
slow rate of phosphorylation of peptide substrate, a particularly
useful system for testing this mechanism. Further studies will be
needed to define the structural changes that attenuate the kinase
through changes in L2's lipoyl group and to determine how the K1
and K2 subunits of the kinase vary in their regulatory control.
In
conclusion, our results strongly support E3-catalyzed
reduction and E2-catalyzed acetylation of lipoyl moiety of the
kinase binding inner (L2) domain of E2 mediating a marked
enhancement in kinase activity. The effectiveness in intact PDC of
acetylating a low proportion of L2 domains in enhancing the
phosphorylation of many bound E1 by a single tightly bound
kinase molecule is probably accomplished by inter-L2 domain movement of
the kinase combined with a stronger interaction of the kinase with
acetylated L2 domains. Several studies on kinase function and
regulation are consistent with a change in a
K-requiring process ultimately occurring in kinase
stimulation. A candidate mechanism involves counteracting
K
-strengthened ADP binding to the kinase to speed up
slow dissociation of this product from the active site of the kinase.