From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229-3900
Received for publication, June 12, 2000, and in revised form, September 29, 2000
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ABSTRACT |
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Yeast mitochondrial
NAD+-specific isocitrate dehydrogenase is an octamer
composed of four each of two nonidentical but related subunits
designated IDH1 and IDH2. IDH2 was previously shown to contain the
catalytic site, whereas IDH1 contributes regulatory properties
including cooperativity with respect to isocitrate and allosteric
activation by AMP. In this study, interactions between IDH1 and IDH2
were detected using the yeast two-hybrid system, but interactions
between identical subunit polypeptides were not detected with this or
other methods. A model for heterodimeric interactions between
the subunits is therefore proposed for this enzyme. A corollary of this
model, based on the three-dimensional structure of the homologous
enzyme from Escherichia coli, is that some interactions
between subunits occur at isocitrate binding sites. Based on this
model, two residues (Lys-183 and Asp-217) in the regulatory IDH1
subunit were predicted to be important in the catalytic site of IDH2.
We found that individually replacing these residues with alanine
results in mutant enzymes that exhibit a drastic reduction in catalysis
both in vitro and in vivo. Also based on this
model, the two analogous residues (Lys-189 and Asp-222) of the
catalytic IDH2 subunit were predicted to contribute to the regulatory
site of IDH1. A K189A substitution in IDH2 was found to produce a
decrease in activation of the enzyme by AMP and a loss of cooperativity
with respect to isocitrate. A D222A substitution in IDH2 produces
similar regulatory defects and a substantial reduction in
Vmax in the absence of AMP. Collectively, these
results suggest that the basic structural/functional unit of yeast
isocitrate dehydrogenase is a heterodimer of IDH1 and IDH2 subunits and
that each subunit contributes to the isocitrate binding site of the other.
NAD+-specific isocitrate dehydrogenase is thought to
be important for regulatory control of mitochondrial energy metabolism primarily because of kinetic responses to adenine nucleotides in
in vitro assays. For example, catalytic activation of this tricarboxylic acid cycle enzyme from Saccharomyces
cerevisiae by AMP (i.e. a response to low levels of
ATP) was described several decades ago (1). A correlate of allosteric
regulation is multisubunit structure, and, based on sedimentation
velocity and gel filtration experiments, an octameric structure was
proposed for yeast isocitrate dehydrogenase (2, 3). However, a
nonequivalence of subunits was suggested by equilibrium binding studies
that demonstrated twice the number of isocitrate binding sites relative
to binding sites for Mg2+ or NAD+ (4).
In more recent studies, the yeast enzyme was shown to be composed of
two nonidentical subunits, IDH1 (Mr = 38,001)
and IDH2 (Mr = 37,755), with both being equally
represented in the holoenzyme (5). The two subunits share 42% identity
at the level of amino acid sequence. Both subunits were shown to be
essential for holoenzyme activity, since disruption of either or both
genes encoding the subunits results in yeast strains that exhibit no
detectable NAD+-specific isocitrate dehydrogenase activity
and that are unable to grow with acetate as a carbon source (6, 7). The
acetate growth phenotype is shared with yeast mutants containing
disruptions in genes encoding other tricarboxylic acid cycle enzymes
including malate dehydrogenase (8) and citrate synthase (9).
The yeast IDH1 and IDH2 subunits also share 32% sequence identity with
Escherichia coli isocitrate dehydrogenase, which requires NADP+ as cofactor. The bacterial enzyme is a homodimer and
contains two identical isocitrate-Mg2+ and
NADP+ binding sites per dimer (12). The E. coli
enzyme exhibits no allosteric regulation but is instead regulated by
phosphorylation. This covalent modification of a specific serine
residue located in the isocitrate binding pocket inactivates the enzyme
under physiological conditions requiring reduced flux through the
tricarboxylic acid cycle (11, 12). The target serine residue of the
E. coli enzyme is conserved in both IDH1 and IDH2, and
mutagenesis was previously performed to determine the function of the
corresponding residues in each yeast subunit (13). An S98A substitution
in IDH2 produced a mutant enzyme that exhibited a 60-fold decrease in
Vmax but retained AMP activation and
cooperativity. In contrast, an S92A substitution in IDH1 produced a
mutant enzyme with primary kinetic defects in regulatory properties of
the enzyme, including loss of both AMP activation and cooperativity
with respect to isocitrate. Based on these results, different functions
were assigned to the subunits, with IDH2 being primarily responsible
for catalysis and with IDH1 playing the primary role in regulation. In
support of these assignments, residues comprising the catalytic site of the bacterial enzyme are more highly conserved in IDH2 than in IDH1
(7). We therefore postulate that both subunits contain isocitrate
binding sites, but while the "active" site in IDH2 is catalytic,
the active site in IDH1 has evolved to bind isocitrate for the
purpose of cooperative control rather than catalysis.
Some subsequent mutagenesis studies have supported these designations
of subunit function (14). In these studies, specific adjacent residues
thought to be important for cofactor specificity were altered. These
changes in IDH2 (D286A plus I287A) resulted in a dramatic decrease in
activity due to a reduction in affinity for NAD+. However,
parallel residue replacements in IDH1 (D279A plus I280A) eliminated AMP
activation of the enzyme. These results supported the designations of
IDH2 as the catalytic subunit and of IDH1 as the regulatory subunit,
and we postulate that homologous adenine nucleotide binding domains
have evolved for binding of the cofactor by IDH2 and for binding of the
allosteric activator by IDH1.
In contrast with results described above, other recent experiments have
revealed that catalytic and regulatory functions are not strictly
confined to distinct subunits. Based on a residue in pig heart
isocitrate dehydrogenase that was previously implicated in the binding
of an adenine nucleotide analog (15), the corresponding conserved
aspartate residues in IDH1 and IDH2 were altered. Mutation of the
conserved aspartate residue (D197A) in IDH2 reduced AMP activation,
cooperativity of substrate binding, and Vmax.
Altering the homologous residue in IDH1 (D191A) produced an inactive
enzyme (14).
To understand the functions of IDH subunits in more detail, the current
study further examines the interactions and specific functions of
subunits in yeast isocitrate dehydrogenase. Based on previous results
showing that both IDH1 and IDH2 subunits copurify when only one is
affinity-tagged (14) and that each residual subunit appears to be
monomeric in the absence of the other subunit (6, 7), we have
investigated the possibility that the basic structural/functional unit
of the enzyme is a heterodimer of an IDH1 and an IDH2 subunit. As a
corollary, based on the bacterial enzyme model for homodimeric subunit
interactions, we have examined the potential contribution of a few
residues from each yeast subunit to the isocitrate binding site of the
other subunit. In light of results presented in this paper, we now
interpret the kinetic effects of the D197A replacement in IDH2 and of
the D191A replacement in IDH1 as indicative of contributions of each
residue to the active site of the other subunit in the heterodimer.
Yeast Strains and Growth Conditions--
In expression
studies, the yeast haploid strain S173-6B (MATa, leu2-3, 112, his3-1, ura 3-57, trp 1-289; Ref. 16) was the parental
wild-type control. Strains containing gene disruptions ( Yeast Two-hybrid Constructs and Analyses--
Polymerase chain
reaction (PCR)1 was used to
synthesize IDH1 and IDH2 gene fragments that
lacked codons for the mitochondrial targeting sequences. PCR primers
also introduced BamHI restriction sites 5' and 3' of the
coding sequences for the mature proteins. The DNA fragments were
subcloned into vectors pGBT9 and pGAD424 (CLONTECH
Laboratories, Inc.), and the inserts were sequenced by the Center for
Advanced DNA Technologies, San Antonio, TX, to ensure that no errors
were introduced by PCR. The BamHI fragments were then
subcloned into two other sets of two-hybrid vectors: pAS2-1 and pACT2
(CLONTECH Laboratories, Inc.) and pGBD-C2 and pGAD-C2 (17). The fusion constructs were then transformed singly and in
all possible binding/activation domain pairs into the appropriate host
strains: the CLONTECH vector constructs into strain
Y190 and the pGBD-C2 and pGAD-C2 fusion constructs into PJ69-4.
Transformants were selected for the presence of plasmids by growth on
YNB glucose plates lacking appropriate nutrients. After restreaking
onto similar plates, transformants were tested for their ability to
activate the reporter gene(s). Y190 transformants were analyzed using a Affinity Purification Tests for Identical Subunit
Interactions--
A 2.3-kilobase pair
XbaI/HindIII fragment containing the
IDH2 gene with codons for a C-terminal histidine tag was
subcloned from pIDH1/IDH2His (14) into pRS316. The
resulting construct (pRS316 IDH2His) was transformed
into the Construction and Purification of Mutant Enzymes--
Mutagenesis
was performed using the Transformer Site-Directed Mutagenesis Kit from
CLONTECH Laboratories and the following primers to
introduce planned substitutions: IDH1 K183A
(5'-CAGCTGTGCATGCCGCAAATATCATG), IDH1 D217A
(5'-CGTCCATCATTGTCGCCAATGCCTCCATGC), IDH2 K189A
(5'-ATTGTGGTACATGCCTCTACTATCCAG), and IDH2 D222A (5'-GAAACTGAA
CTTATTGCCAACAGTGTGTTAAAGG). Another primer was used to
simultaneously eliminate a unique BamHI site in the vector
for selection of the mutant plasmids. All mutations were confirmed by
sequencing. A 1.0-kilobase pair RsrII/EcoRI fragment containing each mutation in IDH1 was subcloned into
pIDH1/IDH2His (14). Similarly, a 1.5-kilobase pair
BglII/HindIII fragment containing each mutation
in IDH2 was subcloned into
pIDH1His/IDH2 (14). Each of the four
resulting plasmids and the two wild-type plasmids
(pIDH1His/IDH2 and
pIDH1/IDH2His) were transformed into a
Kinetic Analyses of Purified Enzymes--
Isocitrate
dehydrogenase activity was measured as described previously (5, 14).
Values for Vmax and S0.5 were
derived from Hanes analysis (21) of initial velocity data. One unit of
isocitrate dehydrogenase activity was defined as production of 1.0 µmol of NADH/min. All assays were performed at 24 °C and initiated
by the addition of isocitrate. Data shown in plots are from a single
representative experiment, whereas tabulated data represent results
from three independent experiments. Hill plots include data points
between 10 and 90% of Vmax.
Yeast Two-hybrid Analyses--
For analysis of subunit
interactions in yeast isocitrate dehydrogenase using the yeast
two-hybrid system, PCR was used to amplify IDH1 and
IDH2 coding regions lacking mitochondrial targeting sequences. The amplified genes were sequenced to ensure that no errors
were introduced by PCR and ligated into three different sets of yeast
two-hybrid plasmids. Two vector pairs, pGBT9 with pGAD424 and pAS2-1
with pACT2, were utilized. The first in each pair contains the
nucleotide sequence for the GAL4 DNA-binding domain, and the second
contains the sequence for the GAL4 transcriptional activation domain.
The reporter gene for protein interaction in the host yeast strain,
Y190, is lacZ with expression under the control of a
GAL4 upstream activation sequence. Transformants of Y190
containing plasmids encoding all possible combinations of GAL4 domain
fusions with IDH1 and IDH2 were isolated, and
Another yeast two-hybrid system with other reporter genes was also used
to test isocitrate dehydrogenase subunit interaction. IDH1
and IDH2 genes were subcloned into pGBD-C2 and pGAD-C2
plasmids. The host strain in this system, PJ69-4A, has expression of
ADE3 and HIS3 genes under the control of two
different GAL4-inducible promoters (17). As shown in Table
I, interaction between IDH1 and IDH2 subunits is indicated by growth on
medium lacking histidine for transformants containing both possible
combinations of constructs. However, transformants containing only one
pair (pGBD-C2IDH2 and pGAD-C2IDH1) indicate
interaction of the two subunits in tests for growth without adenine.
Again, no interaction between identical subunits is detected in
parallel two-hybrid assays. For all two-hybrid systems, each construct
was transformed singly into the appropriate host strain to test for
autonomous activation of the reporter gene. In all cases, these results
were negative.
Thus, interaction between IDH1 and IDH2 was readily detected with
several two-hybrid plasmid pairs. However, the interaction was not
observed in all possible combinations. The reason for this is not
known, but it could be due to conformational constraints introduced by
different fusion proteins. It is clear, however, that identical
subunits (i.e. IDH1 with IDH1 or IDH2 with IDH2) show no
substantial interaction with this method. An additional physical test
for the latter interactions was designed as described below.
Affinity Purification Test for Identical Subunit
Interactions--
Affinity purification was previously used to
demonstrate that IDH1 and IDH2 interactions are significant. Those
studies showed that active holoenzyme could be purified using
Ni2+-NTA column chromatography following the introduction
of five consecutive histidine residues at the carboxyl terminus of
either subunit (14). Here we used a version of this histidine tag
method to further investigate identical subunit interactions. For this, two new yeast strains were created as described under "Experimental Procedures." One strain has the IDH1 gene disrupted and
contains a centromere-based plasmid to provide single copy expression
of IDH2 with a histidine tag; the reciprocal strain has the
IDH2 gene disrupted and expresses a histidine-tagged version
of IDH1 supplied by a plasmid. As a result, each strain has two
versions of the same subunit, one that is native and one that contains a histidine tag. Ni2+-NTA affinity chromatography was
performed using whole cell extracts from each strain. Flow-through,
wash, and eluant fractions were reserved from each column. As shown in
Fig. 1, immunoblot analyses of column
fractions demonstrate that native subunits do not copurify with
otherwise identical affinity-tagged subunits. For example, analysis of
the IDH2 subunits from the
Thus, results from both two-hybrid and affinity purification methods
demonstrate that subunit polypeptides show no substantial homomeric
interactions, at least in the absence of the other subunit, and suggest
that the basic structural unit of the enzyme is a heterodimer of an
IDH1 and an IDH2 subunit.
Mutagenesis and Kinetic Analyses--
Our current structural model
for the yeast enzyme is based on information from the x-ray
crystallographic structure of E. coli
NADP+-dependent isocitrate dehydrogenase (22).
In the homodimeric bacterial enzyme, residues comprising the isocitrate
binding site, as illustrated in Fig. 2,
are predominantly located in one subunit. However, two residues
designated by prime symbols, Lys-230' and Asp-283', are contributed by
the other subunit (Fig. 2). Lys-230' has been shown to be essential for
the decarboxylation step in catalysis (23), and Asp-283' contributes to
coordination of Mg2+ in the active site of the other
subunit (22). These two residues are conserved in similar relative
positions in both IDH1 and IDH2 (Fig. 3).
Based on our model for heterodimeric interaction, we propose that the
conserved residues in IDH1, Lys-183 and Asp-217, might be important in
the catalytic substrate binding site, which is thought mainly to reside
in IDH2. Conversely, the corresponding residues in IDH2, Lys-189 and
Asp-222, could be involved in the regulatory isocitrate binding site
predominantly located in IDH1.
To test these possibilities, we used mutagenesis to independently
introduce alanine codons for IDH1 Lys-183 or Asp-217 codons and for
IDH2 Lys-189 or Asp-222 codons. Mutant alleles for each subunit were
coexpressed with the histidine-tagged allele of the other subunit in a
Measurements of Vmax were conducted using
affinity-purified enzymes. S0.5 values for isocitrate were
measured in the presence or absence of 100 µM AMP, and
corresponding Hill coefficients were calculated. S0.5
values and Hill coefficients for NAD+ were determined in
the presence of AMP. Results of the kinetic studies are illustrated in
Fig. 5 and summarized in Table
II. As previously reported (14), the two
histidine-tagged wild-type enzymes share similar kinetic properties.
S0.5 values for isocitrate are similar and reduced
5-6-fold in the presence of 100 µM AMP. For both
enzymes, Hill coefficients calculated in the presence or absence of AMP
range from 3.2 to 3.9.
As predicted by the model described above, mutant enzymes containing
IDH1 K183A or D217A substitutions (with IDH2 histidine-tagged) exhibit
severe defects in catalytic activity. For these mutant enzymes, using
as much as 19 µg of enzyme per 1-ml reaction produces a barely
measurable change in absorbance unit per min. Thus, the full range of
kinetic parameters for these enzymes could not be determined. However,
based on the minimally measurable activities, the substitution of
alanine for Asp-217 produces a greater catalytic defect than the same
replacement for Lys-183. Respective velocities of 6.7 × 10
As also predicted by the model, we find that the cooperativity with
respect to isocitrate is lost in the mutant IDH2 enzymes containing
K189A or D222A substitutions; i.e. Hill coefficients for
isocitrate are reduced to values slightly greater than 1 (Fig. 5 and
Table II). In addition, allosteric activation of catalytic activity by
AMP is also eliminated by the substitutions for Lys-189 and Asp-222;
i.e. S0.5 values for isocitrate are largely
unaffected by the presence of AMP. Thus, while S0.5 values
for isocitrate measured in the absence of this activator are similar
for mutant and wild-type enzymes, the S0.5 values measured
in the presence of 100 µM AMP are, respectively, 7- and
9-fold greater for the K189A and D222A enzymes than that measured for
the corresponding wild-type enzyme. However, interestingly, AMP does
appear to activate the D222A mutant enzyme by increasing
Vmax approximately 4-fold over values measure in
the absence of AMP. In the absence of AMP, the
Vmax for the D222A mutant enzyme is 18-fold
lower, whereas the Vmax for the IDH2 K189A
enzyme is only 2-fold lower than that of the wild-type control. In
fact, for these assays examining kinetic parameters, it was necessary
to use a concentration of the IDH2 D222A enzyme 10-fold greater than
that of the IDH2 K189A enzyme. Thus, while expected effects on
regulatory properties were obtained with both substitutions, a more
dramatic effect on catalysis is obtained with the D222A substitution.
Relative effects on Vmax are discussed in more
detail below. For both IDH2 K189A and D222A mutant enzymes, apparent
Km values for NAD+ are increased;
however, these effects are relatively minor in comparison with a
70-fold increase previously observed for a mutant enzyme (IDH2
D286A/I287A) with substitutions specifically targeted to alter this
kinetic property (14).
As described above, disruption of IDH1 or IDH2
produces yeast strains unable to grow with acetate as a carbon source
(6, 7). Our original prediction of differential catalytic and regulatory
function for the two homologous subunits of yeast
NAD+-specific isocitrate dehydrogenase was based on
alignment of amino acid sequences and three-dimensional modeling using
the sequence and structure of the E. coli
NADP+-specific enzyme (22). At similar positions in the
sequences and in similar modeled orientations, IDH2 has residues
identical to all of those in the E. coli
isocitrate/Mg2+ binding site illustrated in Fig. 2. IDH1,
on the other hand, shares some identities with residues in the
bacterial enzyme site but differs in four of nine positions (with Ala,
Phe, Thr, and Asn occupying respective residue positions corresponding
to bacterial Arg-129, Tyr-160, Asp-307, and Asp-311). Since IDH1 lacks
two of the bacterial enzyme residues (Asp-307 and Asp-311) involved in
coordination of Mg2+, it seems unlikely that the putative
cooperative site would bind an isocitrate-Mg2+ complex,
potentially explaining previous observations that the yeast enzyme has
twice as many isocitrate as Mg2+ binding sites (4). We are
currently investigating this possibility. This structural information
led us to propose that IDH2 contains the catalytic
isocitrate-Mg2+ binding site and that IDH1 binds isocitrate
alone in a manner important for cooperativity but not for catalysis.
Results of mutagenesis experiments (Refs. 13 and 14; summarized in
Table III) consistent with these
designations of catalytic versus regulatory function include
(a) substitutions for active site serine residues corresponding to bacterial Ser-113 that reduce catalytic activity (IDH2
S98A) versus cooperativity and AMP activation (IDH1 S92A) and (b) substitutions for adjacent residues corresponding to
bacterial residues known to be important for cofactor specificity (25) that eliminate apparent binding of NAD(H) by IDH2 (D286A/I287A) versus of AMP by IDH1 (D279A/I280A).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IDH1::LEU2,
IDH2::HIS3, or
IDH1::LEU2/
IDH2::HIS3)
were constructed using the parental strain as described
previously (6, 7). Yeast strains used in two-hybrid analyses were Y190
(MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901,
leu2-3, 112, gal4
, gal80
, cyhr2,
LYS2::GAL1UAS-HIS3TATA-HIS3,
URA3::GAL1UAS-GAL1TATA- lacZ), provided by CLONTECH Laboratories, Inc., and
PJ69-4 (MAT
, trp1-901, leu2-3, 112, ura3-52,
his3-200, gal4
, gal80
,
LYS2::GAL2- HIS3, GAL2-ADE2,
met2::GAL7-lacZ), provided by Dr. Philip James (17). Strains were grown in YP medium (1% yeast extract, 2% Bacto-peptone) or in YNB medium (0.17% yeast nitrogen base, 0.5% ammonium sulfate, pH 5.8) containing 2% final concentrations of various carbon sources. YNB medium contained the appropriate amino acids or nucleotides required for growth.
-galactosidase colony lift filter assay, which was performed using
CLONTECH Laboratories Protocol PT1030-1. A control
strain contained a plasmid encoding the wild type GAL4 protein. PJ69-4 transformants were tested for their ability to grow on plates in the
absence of adenine or histidine after incubation from 3 to 5 days at
30 °C.
IDH1 host strain. Immunoblot analysis was used to verify that this strain expressed two versions of IDH2, one
native and one histidine-tagged. Similarly, a 4.0-kilobase pair
XbaI fragment containing the IDH1 gene with
codons for a histidine tag was subcloned from
pIDH1His/IDH2 (14) into pRS316. The
resulting construct (pRS316 IDH1His) was transformed
into the
IDH2 strain. This transformant
contained two types of IDH1, one histidine-tagged and one native. The
two strains were grown in 500-ml cultures of YP medium with
glycerol/lactate as the carbon source to induce expression of the
isocitrate dehydrogenase subunits (15). The cells were harvested, and
extracts were used for affinity purification as described previously
(14) with the following modifications. Buffer A contained only 20 mM imidazole, and the column absorption/elution procedure
was not repeated. Column flow-through, wash, and eluant fractions were
combined with equal volumes of loading buffer and electrophoresed on a 15% acrylamide/SDS gel. The gel was transferred to a polyvinylidene difluoride membrane and blocked in 5% bovine serum albumin overnight. For analysis of fractions from the
IDH1
transformant expressing IDH2His, the membrane was incubated
in a 1:500 dilution of IDH antiserum (5), and protein was detected by
autoradiography after incubation of the membrane with
125I-labeled protein A (19). Analysis of fractions from the
IDH2 transformant was conducted using enhanced
chemiluminescence (Amersham Pharmacia Biotech ECL Kit and protocol).
IDH1
IDH2 host strain.
Transformant strains were grown in 0.5-1.0 liters of YP medium with
glycerol/lactate as the carbon source. Affinity-tagged enzymes were
purified by chromatography using Ni2+-NTA resin (Qiagen) as
described previously (14). Concentrations of purified proteins were
determined by the method of Lowry (20). To ensure that holoenzyme was
purified from all strains, samples of ~15 µg of each purified
enzyme were electrophoresed on a 10% acrylamide/SDS gel and stained
with Coomassie Blue (19).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity was determined by a colony filter lift assay. With this assay,
as shown in Table I,
-galactosidase
activity is observed for four of the eight pairs of constructs tested
for interaction between IDH1 and IDH2. Typically, a blue colony color
is detected within 1 h in the positive assays for this
interaction. Positive controls, colonies expressing the full-length
GAL4 protein, typically turn blue in about 30 min with this filter
assay. In contrast, no interaction is detected between identical
subunits expressed with any of these vector pairs (Table I). Negative
results are defined as the absence of detectable
-galactosidase
activity after 10 h.
Yeast two-hybrid assays
IDH1 strain reveals
that only the faster migrating native version of the subunit is found in the column flow-through fraction (Fig. 1A,
lane 1), and neither form of the subunit is
detected in the wash fraction (Fig. 1A, lane
2). Only the higher molecular weight histidine-tagged IDH2 subunit is observed in the eluant from the column (Fig. 1A,
lanes 3 and 4). Similar results are
observed for the
IDH2 strain expressing the
IDH1 subunits, with the bulk of the native subunit and a small portion
of the affinity-tagged species appearing in the column flow-through
fraction (Fig. 1B, lane 1). Again,
only the affinity-tagged form of IDH1 is observed in the fraction
eluted with high concentrations of imidazole (Fig. 1B,
lanes 3-5).
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Fig. 1.
Immunoblot analysis following coexpression
and affinity purification of each native and histidine-tagged IDH
subunit. A, protein extracts from a IDH1 strain
expressing endogenous IDH2 and plasmid-encoded IDH2His were
subjected to Ni2+-NTA column chromatography. Aliquots of
column fractions used for electrophoresis were 100 µl of flow-through
(lane 1), 100 µl of wash (lane
2), 6 µl of eluant (lane 3), and 12 µl of eluant (lane 4). B, protein
extracts from a
IDH2 strain expressing native IDH1 and
plasmid-encoded IDH1His were subjected to
Ni2+-NTA column chromatography. Aliquots of column
fractions used for electrophoresis were 80 µl of flow-through
(lane 1), 80 µl of wash (lane
2), and increasing volumes of eluant (40 µl
(lane 3), 50 µl (lane 4),
and 60 µl (lane 5)). Conditions for
electrophoretic resolution of the faster migrating native form and the
histidine-tagged form of each subunit are described under
"Experimental Procedures."
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Fig. 2.
Representation of the
Mg2+-isocitrate binding site of E. coli
isocitrate dehydrogenase as described by Hurley et
al. (22). Prime residues Lys-230' and
Asp-283' are contributed by the other subunit in the bacterial
homodimer. Below are listed residues of yeast IDH1 and IDH2
subunits predicted from sequence and structural alignments to
correspond with each residue in the bacterial catalytic site.
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Fig. 3.
Partial amino acid sequence alignments of
IDH1 and IDH2 with E. coli isocitrate
dehydrogenase. Boxed are bacterial residues Lys-230'
and Asp-283' that contribute to the isocitrate binding site of the
other subunit. Conserved residues are shown in boldface
type.
IDH1
IDH2 yeast strain. Mutant
enzymes were purified by Ni2+-NTA affinity chromatography
as described under "Experimental Procedures." Samples of all
purified enzymes were electrophoresed on a denaturing gel and stained
with Coomassie Blue. As shown in Fig. 4,
both subunits copurify during affinity chromatography of the mutant
enzymes. Differences in electrophoretic mobility are due to the
histidine tags (e.g. compare mobility of the more slowly
migrating histidine-tagged form of IDH1 in lanes
1-3 versus that for the native IDH1
subunit in lanes 4 and 5) and
presumably to amino acid substitutions in mutant subunits as also
previously observed (14). Differences in mobility are observed for both subunits containing substitutions for aspartate residues, D222A in IDH2
(Fig. 4, lane 2) and D217A in IDH1 (Fig. 4,
lane 5). None of the residue substitutions
appears to have any gross conformational effect on holoenzyme
structure, since both subunits copurify when only one subunit is
affinity-tagged.
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Fig. 4.
Electrophoretic analysis of affinity-purified
wild-type and mutant forms of yeast isocitrate dehydrogenases.
Histidine-tagged enzymes were expressed and purified as described under
"Experimental Procedures." Samples containing ~15 µg of each
purified enzyme were utilized for electrophoresis and Coomassie Blue
staining. The purified enzymes include IDH1His/IDH2
(lane 1),
IDH1His/IDH2D222A (lane
2), IDH1His/IDH2K189A
(lane 3), IDH1/IDH2His
(lane 4),
IDH1D217A/IDH2His (lane
5), and IDH1K183A/IDH2His
(lane 6). In each lane, the slower
migrating band is IDH1, and the faster migrating band is IDH2.
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Fig. 5.
Kinetic analyses of wild-type and mutant
forms of isocitrate dehydrogenases. Velocity saturation curves for
D-isocitrate are shown for enzymes in the absence
(closed symbols) or presence (open
symbols) of 100 µM AMP. Data for both
wild-type enzymes (IDH1His/IDH2 (circles) and
IDH1/IDH2His (triangles)) are shown in the top
panel. The insets are Hill plots of the same
data.
Kinetic properties of affinity-purified enzymes
3 units/mg and 3.2 × 10
2 units/mg are more than 1000- and 300-fold
lower than the Vmax value of the corresponding
wild-type enzyme.
IDH1
IDH2 yeast strains
expressing the mutant enzymes constructed for this study were tested
for growth in medium containing acetate to determine whether the mutant
enzymes could compensate for the loss of wild type isocitrate
dehydrogenase. Strains containing the IDH1 K183A or D217A enzyme were
found to be unable to grow with acetate as a carbon source, suggesting
that these enzymes also lack significant catalytic activity in
vivo. In contrast, expression of the IDH2 K189A or D222A enzyme is
able to restore growth in acetate medium with generation times (~4.5
h) comparable with those measured for the parental wild-type strain.
Thus, despite the loss of regulatory properties and the decreased
activity of the IDH2 D222A enzyme, these mutant enzymes can apparently
replace the wild-type enzyme during steady-state growth on acetate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Summary of kinetic effects of reciprocal residue replacements in IDH
subunits
Results from both yeast two-hybrid assays and affinity chromatography (Ref. 14; this study) indicate that there is significant physical interaction between IDH1 and IDH2 subunits, whereas identical subunit polypeptides show no substantial interactions with these tests. This experimental evidence suggests that the basic structural/functional unit of this enzyme is a heterodimer, although other interactions clearly occur within the octameric holoenzyme. Furthermore, based on the structure of the E. coli isocitrate dehydrogenase homodimer, which shows that specific residues, Lys-230' and Asp-283' (Fig. 2), from one subunit contribute to the isocitrate binding site of the other, results of this study suggest that the same is true for the nonidentical subunits of the yeast enzyme, i.e. that reciprocal contributions to the active site in each subunit are made by a few residues from the other polypeptide chain. The essential nature of these reciprocal interactions would explain the absence of catalytic activity for IDH2 alone despite the apparent presence of all necessary catalytic residues in this subunit. In other words, Lys-189' and Asp-222' residues of IDH2 apparently do not function to complete homomeric catalytic active sites and instead contribute to the heteromeric cooperative active site of IDH1. Reciprocally, the Lys-183' and Asp-217' residues of IDH1 appear to be essential components of the IDH2 catalytic site.
Previous and current kinetic studies of mutant enzymes summarized in Table III support these hypotheses. Activity is dramatically reduced with alanine substitutions for residues with putative catalytic functions in substrate isocitrate binding (IDH2 Ser-98 (13) and IDH1 Lys-183' or Asp-217' (this study)) or in cofactor NAD(H) binding (IDH2 Asp-286/Ile-287 (14)). For these catalytic mutant enzymes, when kinetic properties are measurable, there is no deleterious effect on cooperativity or activation by AMP. We believe that the IDH1 mutant enzyme with an alanine substitution for Asp-191 (14) also belongs in this category. As described above, this residue was originally targeted for mutagenesis based on correspondence with an aspartate residue in the pig heart enzyme that is selectively modified by an adenine nucleotide analogue (15). Although this residue is not part of our model for the active sites, the kinetic properties of the IDH1 D191A enzyme are consistent with its designation as Asp-191', i.e. with a function in the catalytic site of IDH2. This residue is located near IDH1 Lys-183', and it corresponds to the bacterial residue Asp-238 located near residues 234-236 that are known to be involved in intersubunit contacts (24).
For mutant yeast enzymes with substitutions in residues with putative functions in regulatory binding of isocitrate (Table III; IDH1 Ser-92 (13) and IDH2 Lys-189' or Asp-222' (this study)), both cooperativity and reduction of S0.5 values by AMP are defective. Adjacent substitutions within the putative AMP binding region of IDH1 (Asp-279/Ile-280 (14)) produce a loss of activation but no effect on cooperativity. We also include in this regulatory category a previous mutant enzyme with a substitution for IDH2 Asp-197' (14), the reciprocal of IDH1 Asp-191'. This residue is located near IDH2 Lys-189', and the IDH2 D197A enzyme exhibits kinetic defects consistent with a contribution to the regulatory isocitrate binding site of IDH1.
Of particular interest are the effects of substitutions for "regulatory" residues on Vmax (Table III). None of these effects are as striking as the defects produced with substitutions for "catalytic" residues, but deleterious effects range from 2- to 18-fold decreases relative to Vmax values for corresponding wild-type enzymes. That we always see some effect on catalysis indicates that full catalytic activity may require regulatory functions. It was in fact predicted over 30 years ago (1) that either the regulatory isocitrate site or the AMP binding site must be occupied to obtain maximal catalytic activity. However, because effects on Vmax vary significantly for mutant enzymes that exhibit similar defects in cooperativity, it may be that some residues have dual functions or that portions of the polypeptide chains containing these residues may be located in proximity of both subunit active sites. IDH2 Asp-222' is particularly interesting in this respect. This residue appears to be important for regulatory properties presumably through contributions to the IDH1 active site. However, its replacement also has a deleterious effect on Vmax, more so in the absence of AMP (an 18-fold decrease) than in the presence of AMP (a 4-fold decrease). One possibility is that IDH2 Asp-222' is important for functional communication between the active sites of the IDH1 and IDH2 subunits, particularly in the absence of allosteric activator.
Overall, it appears that the yeast IDH1 and IDH2 subunits have
independently evolved for different functions but that physical interactions between the subunits, including residues that contribute to reciprocal subunit function, have been maintained. It is also clear
that, while the bacterial and yeast isocitrate dehydrogenases differ
substantially in terms of subunit composition, cofactor specificity,
and regulation, significant structural motifs have been conserved. We
predict that information obtained from the E. coli enzyme
will continue to aid in functional analysis of the more complex
S. cerevisiae homologue. Ongoing crystallographic analysis
of the yeast enzyme should also clarify, by comparison with the
bacterial structure, the basis and evolution of its allosteric properties.
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ACKNOWLEDGEMENTS |
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We thank Dr. Wen-Ning Zhao for construction of the IDH1 K183A and IDH2 K189A mutant enzymes. We also appreciate the kinetic troubleshooting expertise of Dr. Steve Ingram and assistance in kinetic analyses from Dr. Larry D. Barnes.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM51265.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 210-567-3782;
Fax: 210-567-6595; E-mail: henn@uthscsa.edu.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M005056200
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; NTA, nitrilotriacetic acid.
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