From the Department of Biology, Boston
University, Boston, Massachusetts 02215 and the § Department
of Physiology and Biophysics, Boston University School of Medicine,
Boston, Massachusetts 02118-2394
Received for publication, September 9, 2002, and in revised form, February 27, 2003
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ABSTRACT |
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Vertebrate fructose-1,6-bisphosphate
aldolase exists as three isozymes (A, B, and C) that demonstrate
kinetic properties that are consistent with their physiological role
and tissue-specific expression. The isozymes demonstrate specific
substrate cleavage efficiencies along with differences in the ability
to interact with other proteins; however, it is unknown how these
differences are conferred. An alignment of 21 known vertebrate aldolase
sequences was used to identify all of the amino acids that are specific to each isozyme, or isozyme-specific residues (ISRs). The location of
ISRs on the tertiary and quaternary structures of aldolase reveals that
ISRs are found largely on the surface (24 out of 27) and are all
outside of hydrogen bonding distance to any active site residue.
Moreover, ISRs cluster into two patches on the surface of aldolase
with one of these patches, the terminal surface patch, overlapping with the actin-binding site of aldolase A and overlapping an area of higher than average temperature factors derived from the
x-ray crystal structures of the isozymes. The other patch, the distal
surface patch, comprises an area with a different electrostatic surface
potential when comparing isozymes. Despite their location distal to
the active site, swapping ISRs between aldolase A and B by multiple
site mutagenesis on recombinant expression plasmids is sufficient to
convert the kinetic properties of aldolase A to those of aldolase B. This implies that ISRs influence catalysis via changes that alter the
structure of the active site from a distance or via changes that alter
the interaction of the mobile C-terminal portion with the active site.
The methods used in the identification and analysis of ISRs discussed
here can be applied to other protein families to reveal functionally
relevant residue clusters not accessible by conventional primary
sequence alignment methods.
Vertebrate fructose-1,6-bisphosphate aldolase is a ubiquitous
tetrameric enzyme that catalyzes reactions in the glycolytic, gluconeogenic, and fructose metabolic pathways (1). The enzyme catalyzes the reversible aldol cleavage of Fru
1,6-P21 into two
trioses, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. It also cleaves the structurally related sugar Fru 1-P
into glyceraldehyde and dihydroxyacetone phosphate (2, 3).
Vertebrate aldolases exist as three or more isozymes with different
tissue distributions: aldolase A (expressed predominantly in muscle),
aldolase B (expressed predominantly in liver), and aldolase C
(expressed predominantly in brain) (1). The sequences of the mammalian
isozymes are highly conserved, exhibiting 81% sequence identity
between aldolases A and C (4). Aldolase B is slightly more divergent
with ~70% sequence identity to both aldolases A and C (5).
Consistent with this sequence similarity, isozymes A and C exhibit
comparable kinetic properties. Aldolases A and C have evolved to
perform the glycolytic reaction, Fru 1,6-P2 cleavage, more
efficiently than aldolase B as demonstrated by a 20-30-fold higher
kcat (6, 7). Conversely, aldolase B cleaves Fru
1-P three times more efficiently than aldolase A or C (6). In addition,
aldolases B and C demonstrate a 10-fold lower Km for
glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, implying
that they have evolved to perform the gluconeogenic reaction, Fru
1,6-P2 synthesis, more efficiently than aldolase A (8).
Although these kinetic properties are consistent with their
physiological roles and tissue-specific expression (9), the structural
features that confer these isozyme-specific differences are unknown.
The three-dimensional structures of aldolase A (10) and B (11) have
been determined and reveal homotetramers of very similar Given that aldolase isozymes have evolved distinct kinetic properties,
residues unique to each isozyme must be conserved. Such
isozyme-specific residues (ISRs) for aldolases B and C have been
identified previously (17, 18). The ISRs were identified based on the
criteria that the residue is conserved within orthologs and is distinct
among paralogs. In this report, we combined this evolutionary
information with the three-dimensional structures of aldolase A, B, and
a model of C to identify structural features that may be involved in
conferring isozyme-specific kinetic preferences. To this end, an
alignment of all six vertebrate aldolase A sequences was performed to
identify aldolase A ISRs, along with a more extensive alignment of
aldolase B and C sequences. All three sets of ISRs were mapped onto the
conserved three-dimensional structure of the enzyme, and two patches on
the surface of aldolase, where ISRs from all three isozymes cluster,
were identified. The terminal surface patch (TSP) was associated with
an area of relatively high flexibility as demonstrated by high x-ray
crystallographic temperature factors. In addition, the F-actin-binding
domain overlapped with this patch; thus, isozyme-specific surface
moieties were correlated with macromolecular interactions.
ISR clusters on the surface of aldolase may confer kinetic specificity
by influencing the active site directly via the C-terminal region or
indirectly through conformational changes that alter the structure of
the active site from a distance. To test this hypothesis, PCR-assisted
multiple site-directed mutagenesis was used to incorporate all 12 aldolase B ISRs into the appropriate position of aldolase A while at
the same time replacing the 11 aldolase A ISRs with the analogous
residue of aldolase B. The resulting protein was expressed in
Escherichia coli and purified. Kinetic constants were
determined, and a direct correlation between kinetic constants and ISR
type was found. This type of analysis of ISRs can easily be applied to
other protein families to reveal functional residue clusters not
apparent from primary sequence alignments.
Materials--
Restriction endonucleases, T4 DNA ligase,
exonuclease III, and DNA polymerase I were from New England Biolabs.
Calf intestine alkaline phosphatase and glycerol-3-phosphate
dehydrogenase/triose phosphate isomerase were from Roche Applied
Science. Deoxynucleoside triphosphates, CM-Sepharose®
CL-6B, and Sephadex® G-150 were from Amersham Biosciences.
Oligonucleotides used for site-directed mutagenesis and sequencing were
either synthesized on DNA synthesizers using phosphoramidite chemistry
and the manufacturer's protocols or purchased from The Midland
Certified Reagent Company, Inc. Ultrapure sucrose was from Invitrogen.
Fru 1,6-P2, triethanolamine, and other chemicals were from Sigma.
Identification of Isozyme-specific Residues--
The amino acid
sequences of the following aldolase A enzymes were aligned:
human (19), mouse (20), rabbit (21), rat (22),
chicken,2 and
Xenopus (23). The alignment was generated using Clustal V
(24). Amino acids that are unique to each isozyme and distinct from the
analogous amino acid in paralogs were identified and defined as ISRs.
This comparison included the addition of mouse B (25) and C (26),
salmon B (27), and pufferfish
C3 to the previously
identified ISRs (17, 18). The presence of non-conservative (radical)
and conservative differences between isozyme groups were evaluated
using the three-dimensional structure of aldolase A (Protein
Data Bank (PDB) accession code 1ADO) (13) and the graphics program O
(28) to assess whether the ISR played a crucial role in stabilizing
tertiary or quaternary structure. These determinations were based on
the degree of structural alteration caused by each substitution,
location of the residue in the three-dimensional structure of the
aldolase tetramer, polarity, charge, size, and aromaticity (29).
Conservative changes included residues that were located in a
structural motif such as an Site-directed Mutagenesis, Expression, and Purification of
Recombinant Aldolase--
The H156E enzyme was constructed via
site-directed mutagenesis (30) on the M13-based cDNA clone, AM1
(31). The H156E substitution was generated using the
oligodeoxyribonucleotide: 5'-GATTGGGGAAGAGACCCCCTCAG-3'. Underlined nucleotides refer to the codon that has been changed from
that of the wild-type aldolase A. The mutant enzyme AB_All was
generated via multiple site-directed mutagenesis using overlapping oligodeoxyribonucleotides (32) that complement the high copy ATG vector
expressing rabbit aldolase A, pPB14 (33). This construction involved
three steps. First, the mutant expression plasmids, termed pABN and
pABC, were generated. The following oligodeoxyribonucleotides were used
to construct pABN: geneU, 5'-ACGACCGAGCGCAGCGAGTCAGTGAG-3' (forward);
ABN1, 5'-GCAGCCTCTTCCCCATGGTCCCGGTCGACTCATC-3' (reverse); ABN2,
5'-CGACCGGGACCATGGGGAAGAGGCTGCAACGCATCGGTACC-3'
(forward); ABN3,
5'-CACGCTGTCATCCGCGGTCAGCAGCAGCTCCCGGAAGAAACGCCGGTTCTC-3' (reverse); ABN4,
5'-GCTGCTGACCGCGGATGACAGCGTGAACCAGTGCATCGGGGGC GTCATC-3'
(forward); ABN5, 5'-GCCCACAACAATGCCCTTGGACTTGAT-3'
(reverse); ABN6, 5'-CAAGTCCAAGGGCATTGTTGTGGGCATCAAG-3'
(forward); ABN7, 5'-CCAGACAGGAAGCAAACTCCAGTGACAG-3' (reverse); ABN8, 5'-CACTGGAGTTTGCTTCCTGTCTGGAGGTCAG-3'
(forward); ABN9, 5'-AGGAGAAGGTCAGTTTCCACGGCTTCAGCAG-3'
(reverse); ABN10, 5'-GCTGAAGCCGTGGAAACTGACCTTCTCC-3'
(forward); ABN11,
5'-CACGAACTCCTCCTGGGCAGCCTTCTTGTTCTCCTTCTTCCCACCCCAGGCCGCCAGAGCCGAGGC-3' (reverse); ABN12,
5'-GAAGGCTGCCCAGGAGGAGTTCGTGAAGCGGGCCATGGCCAACAGCCTCGCATGCCAAGGGAAGTAC-3' (forward); geneL, 5'-GCTACTGCCGCCAGGCAAACTGTTTTATCAG-3' (reverse). The
following oligodeoxyribonucleotides were used to construct pABC:
geneU (see above) (forward); ABC1,
5'-GGGTGGCCGCGGCCCCGGCCTGACCACTCGGGACGTACTTCCCTTGGCATGCGAGGCTGTTG-3' (reverse); ABC2,
5'-GGTCAGGCCGGGGCCGCGGCCACCCAGTCCCTCTTCACCGCTAACTACGCCTACTAAG-3' (forward); geneL (see above) (reverse). The boldface
nucleotides in oligodeoxyribonucleotides ABN12 and ABC1 represent a
silent mutation engineered to include a unique SphI
restriction site. This SphI site, along with an upstream
EcoRI and a downstream HindIII site, was used to
splice together pABN and pABC to generate pAB. All of the ISRs were not
included in pAB, and some residues were included that were later
determined not to be ISRs after the addition of recently published
aldolase sequences (see "Results"). The construct pAB_All was
generated from pAB using the oligodeoxyribonucleotides: geneU (see
above) (forward); AB_All1,
5'-GCACTGGTTCACGCGTCATCCGCGGTCAG-3' (reverse);
AB_All2, 5'-GACCGCGGATGACCGCGTGAACCAGTG-3' (forward); AB_All3,
5'-GCCCACAACAATGCCCTTGGACTTGATAACTTGCGGGAAGGGCTTCCCATCGTC-3' (reverse); AB_All4,
5'-ATCAAGTCCAAGGGCATTGTTGTGGGCATCAAGGTAGACAAGGGCGTGGCACCTCTGG-3' (forward); AB_All5,
5'-GGCGAGGGCTGAGGGGGTCTGTTCCCCAATCTTC-3' (reverse); AB_All6, 5'-GGGGAACAGACCCCCTCAGCCCTCGCCATCATG-3' (forward);
AB_All7, 5'-CCACAATGGGCACAAGGCCATTCTGCTGGCAGATG-3'
(reverse); AB_All8, 5'-GCCAGCAGAATGGCCTTGTGCCCATTGTG-3' (forward);
AB_All9,
5'-CCAGACAGGAAGGTAACTCCAGTGACAGCGGCGGGCACTGTG-3' (reverse); AB_All10,
5'-GCCCGCCGCTGTCACTGGAGTTACCTTCCTGTCTG-3' (forward); AB_All11,
5'-ACTTCCCTTGGCATGCGAGGCTGTTGGCCAGGGCCCGCTTCAC-3' (reverse);
AB_All12,
5'-GGCCAACAGCCTCGCATGCCAAGGGAAGTACACCCCGAGTGGTCAG-3' (forward); geneL (see above) (reverse). The codons in italics indicate sites where pAB was reverted back to wild type. The
amplification of each fragment was performed by incubating at 94 °C
for 1 min followed by 30 cycles at 94 °C for 30 s, 60 °C for
30 s, and 72 °C for 1 min. The overlapping fragments
were generated with 35 cycles. The resulting missense mutations and
their consequent amino acid substitutions are shown in Table
I. Potential mutants were screened by DNA
sequence determination using dideoxy termination (34). The expression
plasmid pXPB (33) was used for the expression of human aldolase B. The
expression and purification were performed as described previously (31)
using an affinity elution from CM-Sepharose CL-6B with Fru
1,6-P2 at pH 7.0 for aldolase A and B, at pH 7.6 for H156E,
and at pH 8.3 for AB_All.
Characterization of Mutant Aldolases--
Activity of Fru
1,6-P2 and Fru 1-P cleavage was measured as described
previously (31) by the decrease in A340 in an
assay coupled to NADH oxidation by glycerol-3-phosphate dehydrogenase (35). Sucrose density gradient sedimentation velocity was determined for the H156E mutant using a modified procedure of Martin and Ames (36) as described previously (37). Briefly, 0.1 mg of sample was loaded on 5-20% sucrose gradients at 20 °C and
sedimented for 14.5 h at 38,000 rpm in a SW60 rotor
( Construction of Aldolase C Homology Model--
The model of
aldolase C was derived from the high resolution structure of aldolase A
(PDB accession code 1ADO) (13) using the Swiss-Model protein modeling
server (38). Comparisons of amino acid and nucleotide sequences have
shown that aldolase C and aldolase A are more closely related to each
other than either is to aldolase B (4). The protein modeling server
performs superposition of related tertiary structures, generation of a multiple sequence alignment with the sequence to be modeled, generation of a structural framework of the new sequence, rebuilding of missing loops, completion and correction of backbone and side-chain geometry, and refinement of the structure by energy minimization and molecular dynamics.
Three-dimensional Mapping of ISRs--
The locations of ISRs
were mapped to the tertiary and quaternary structure of aldolase A
using the program O (28). The coordinates of aldolase A and B were
taken from high resolution crystal structures (PDB accession codes 1ADO
and 1QO5, respectively) (11, 13), and those of aldolase C were
generated from the homology model (see above). The electrostatic
potential surfaces of aldolase A, B, and C were calculated and
displayed using the program GRASP (39).
Identification of Aldolase A ISRs--
The amino acid sequences of
21 vertebrate aldolase isozymes were analyzed to identify individual
residues specific to each isozyme. Multiple sequence alignment revealed
a total of 11 aldolase A ISRs (Fig. 1).
The alignment of these aldolase A ISRs was compared in order to further
categorize these isozyme-specific differences into either conservative
or non-conservative (radical) amino acid substitutions in an effort to
identify which substitutions are more likely to confer functional
changes to the isozyme. From this analysis, it was determined that
among the aldolase A ISRs, Ile-39, His-156, Tyr-327, and Ser-353 were
conservative differences and that Ser-45, Pro-71, Arg-91, Gly-102,
Lys-311, Glu-354, and Ser-359 were non-conservative differences with
respect to the other isozymes (Fig. 1). Due to the lack of a definitive
structure, the ISRs in the C-terminal region, Ser-353, Glu-354, and
Ser-359, were categorized without consideration of the
three-dimensional location.
Aldolase A ISRs were compared with the aldolase B (17) and aldolase C
ISRs (18). The inclusion of mouse and salmon aldolase B sequences in
the aldolase B alignment revealed that the previously identified
residues Ser-68 and Cys-268 are no longer conserved (Pro-68 and Thr-268
in salmon) and were thus excluded from the aldolase B ISRs. Similarly,
the inclusion of the pufferfish aldolase C sequence revealed that the
previously identified aldolase C ISR, Leu-57, was not conserved (Arg-57
in pufferfish), and therefore this residue was not included among the
aldolase C ISRs. As a result, a total of 27 ISRs were identified: 11 aldolase A ISRs, 12 aldolase B ISRs, and 4 aldolase C ISRs (Fig. 1).
Notably, the aldolase C ISRs were found within the last 49 residues of
the protein sequence, whereas aldolase A- and B-specific residues were
found throughout the primary sequence.
Locations of ISRs in the Three-dimensional Structure--
The
conservation of ISRs among orthologs implies that they play a role in
conferring the specific kinetic properties of each isozyme. Thus, the
expectation was that they would map largely at or near the active site.
More specifically, the expectation was that they might reside within
hydrogen bonding distance (2.6-4.0 Å) of either Lys-229 or Asp-33,
which are residues known to be involved in catalysis (12). The
locations of all 27 ISRs in three-dimensional space were examined by
mapping the residues onto the high resolution crystal structure of
rabbit aldolase A (PDB accession code 1ADO) (13), which includes
several conformations for the C terminus (residues 345-363). After
analyzing the C termini of the four different monomer structures, the
structure with the C terminus closest to the
To determine whether the ISRs were involved in the subunit-subunit
interactions that might stabilize the tetramer, the locations of ISRs
were examined with regard to the tetramer structure. Only one ISR,
His-156 of aldolase A, was located at the subunit interfaces and was
near the 2-fold symmetry axis of the dimers. Its location implies that
His-156 may play a role in stabilization of the tetramer. The role of
this residue was examined by site-directed mutagenesis. His-156 was
mutated to a glutamic acid in an attempt to reverse the point charge of
this residue. The purified aldolase A H156E mutant enzyme demonstrated
similar kinetics (Table II) and stability to the wild-type enzyme. The kinetic constants for recombinant wild-type aldolase A and B were obtained in the same fashion as for the
mutant enzymes, and the kinetic constants agree with the published data
(Table II). Comparison of the H156E far ultraviolet circular dichroism
spectrum with that of wild-type aldolase A did not reveal any
differences in secondary structure. Gel filtration analysis
demonstrated a quaternary structure similar to wild type. In addition,
thermal stability, as assessed by activity, was similar to that of
wild-type enzyme (data not shown). Thus, there was no evidence
supporting the stabilization of quaternary structure by ISR
His-156.
Remarkably, the majority of ISRs (24 out of 27) were located on the
surface of the protein and generally cluster in two patches, regardless
of the particular isozyme (Fig. 2b). The other 3 ISRs, 58, 182, and 327, were buried in the ISRs Impact on Enzymatic Function--
Although the majority of
ISRs cluster on the surface of aldolase, one model is that these
residues influence kinetic properties at a distance from the active
site. Long distance effects have been documented in other enzymes, such
as trypsin (40) and lactate dehydrogenase (41). The complete set of
ISRs from one aldolase isozyme incorporated into the primary sequence
of another isozyme would test this model and verify the importance of
these ISRs. At the same time, the ISRs of the latter isozyme need to be
removed. Specifically in this study, a mutant enzyme was engineered by swapping the ISRs from aldolase B into aldolase A, whereas at the same
time exchanging the aldolase A ISRs with the analogous residue from
aldolase B (Table I). Due to the fact that no structure is available
for aldolase C and the kinetic differences are greater between
aldolases A and B, the ISRs of only these two isozymes were swapped. To
accomplish this, PCR-assisted multiple site-directed mutagenesis was
used to engineer 32 nucleotide substitutions in an expression plasmid
for aldolase A (Table I). It should be noted that even with the number
of substitutions, the resulting enzyme, AB_All, named in reference to
the swapping of all ISRs, was 93% identical to aldolase A and only
75% identical to aldolase B. After expression and purification of
AB_All, the steady-state kinetics toward the substrates Fru
1,6-P2 and Fru 1-P were determined using an enzyme-coupled
assay (35). Comparing these data with data obtained for isozymes A and
B revealed that both Km and
kcat values toward Fru 1,6-P2
decreased significantly from those of aldolase A and are nearly
identical to that of aldolase B (Table II). Similarly, the
Km of AB_All toward Fru 1-P decreased, whereas the
kcat increased appropriately to mimic the
kinetic parameters of aldolase B (Table II). These results verify that
the set of ISRs identified here is necessary and sufficient for
determining isozyme-specific kinetic properties and suggest that some
of these residues impact their effects at a distance from the active
site. In addition, the fact that the kcat toward the substrate Fru 1-P is increased relative to the parent protein demonstrates that we have not simply generated a mutant with impaired function.
There are two ways in which these apparent kinetic effects could occur
from a distance. First, these residues may act as structural modulators, modifying the structure of the active site from a distance
through conformational effects as in trypsin (40) and lactate
dehydrogenase (41). Second, the residues may act through direct contact
with the active site via the mobile C-terminal region. For example, in
the case of the TSP, a conformational change could position a portion
of the TSP proximal to the active site. It has been hypothesized that
the C-terminal region of aldolase is involved in catalysis through a
possible conformational change coordinated with substrate cleavage (10,
12, 14). One piece of evidence for this involvement comes from previous
studies involving a recombinant chimera of human aldolase B (15). The
section of primary sequence exchanged between aldolase A and B included 17 ISRs identified in this study, encompassing all the TSP residues.
The prediction of functionally important residues in proteins has been
investigated previously using more generalized methods (42-45).
Recently, three-dimensional cluster analysis was used to identify
functionally relevant residue clusters of aldolase (46). Patches of
conserved residues, which could not be identified previously by
sequence alignment and phylogenetic analysis, included the highly
conserved active site and subunit interfaces along with a patch near
the C-terminal region and another that overlapped with the
actin-binding region. The C-terminal and actin-binding patches they
identified overlap with the TSP discussed here. Mutations in the
C-terminal portion implicate this region in conferring isozyme
specificity (14-16, 46, 47). However, an interpretation of the
mechanism by which these residues confer specificity cannot be
substantiated without the definitive location and functional assessment
within this apparently flexible region.
Potential Role of ISRs in Protein-Protein Interactions--
The
binding region on aldolase A by F-actin has been identified by peptide
mapping with a synthetic peptide corresponding to the aldolase sequence
32-52 (48) and by site-directed mutagenesis of binding residues
Arg-42, Lys-107, and Arg-148 (49). This actin-binding site overlaps
with the TSP, which includes ISRs 38, 39, 40, and 45. The residue
Arg-42 is required for actin binding (49) and is located in the center
of the TSP. This residue is conserved in all aldolase isozymes, which
is consistent with the finding that all three isozymes bind to the
cytoskeleton (50). Although all isozymes have the ability to bind to
the cytoskeleton, the interaction is preferable for cytoskeleton
derived from the same tissue from which the isozyme is expressed.
Aldolase A binds most tightly to cytoskeleton isolated from muscle
tissue, and aldolase B binds most tightly to cytoskeleton isolated from
liver tissue. Aldolase C, however, shows only slight preference
for liver- and brain-isolated cytoskeleton (50). These results, together with the fact that the actin-binding site overlaps with the
TSP, suggest that, although Arg-42 is required for binding actin, the
surrounding ISRs confer preference for tissue-specific actin.
The other ISRs that do not overlap with the F-actin-binding site are
also located on the surface. This suggests that they may participate in
tissue-specific protein-protein interactions. Orthologous conservation
of these surface patches reveals potential sites of protein-protein
interactions and implies that isozyme specificity is mediated through
surface interactions. In addition to F-actin (49), the aldolase A
binding partners include vacuolar H+-ATPase (51) and GLUT4
(52). The interaction between aldolase A and F-actin or GLUT4 can be
disrupted by substrates of aldolase, thus implicating structural
connectivity of binding residues with residues of the active site (52).
Aldolase B has been shown to interact with vacuolar
H+-ATPase (51) and the hepatocyte matrix (53). Aldolase C
has demonstrated binding to inositol 1,4,5-trisphosphate (54) and S-100
A1 (55) protein, thus implicating aldolase C in calcium signaling. Such
protein-protein interaction with proteins not involved in glucose
metabolism suggests alternate cellular functions for aldolase, which
may be isozyme-specific.
The method of mapping evolutionarily conserved residues on the tertiary
structure reveals interesting structural attributes that might
correlate with protein-protein interactions. A correlation between
elevated temperature factors derived from x-ray crystal structures and
sites of protein-protein interactions is apparent from studies of
proteases and their inhibitors (56). Several proteases have
demonstrated a preference for cleavage at relatively flexible areas of
a protein substrate as defined by crystallographic temperature factors.
In this study, support for the role of ISRs in protein-protein
interactions is suggested by the relatively high temperature factors of
these residues as compared with the other residues in the crystal
structure. The average temperature factor for all residues in the
aldolase A structure (13) is 21.6 Å2, whereas the average
temperature factor for the ISRs is 37.7 Å2. Similarly, all
residues in the aldolase B structure (11) have an average temperature
factor of 40.0 Å2, whereas the ISRs average 50.9 Å2. The elevated temperature factor among ISRs indicates
that they are located in an area of relative flexibility. Moreover,
this flexibility cannot be attributed solely to the C-terminal region as the majority of ISRs not located in this region also demonstrate elevated temperature factors. Mapping of temperature factors on the
three-dimensional structure reveals that the TSP and the actin-binding site overlap with an area of flexibility in both aldolase A (Fig. 3a) and B (Fig.
3b). The average temperature factors for the ISRs that
comprise the TSP are 58.1 and 62.8 Å2 in the aldolase A
and B structures, respectively.
If each isozyme interacts with specific proteins in tissues where they
are expressed via ISR interactions, they might show different
electrostatic surface potentials at these putative binding sites. To
test this idea, three-dimensional atomic structures of aldolase A and B
and a homology model of aldolase C were used to calculate the
electrostatic surface potentials in the area of the mapped ISR patches
(Fig. 4). The electrostatic surface of
each isozyme in the DSP was indeed distinct between the three isozymes,
whereas they were similar in the TSP. Other areas of the surface were
also similar among the isozymes. Despite its higher overall sequence
identity, the electrostatic surface of the DSP of aldolase C was the
most negatively charged, in contrast to those of aldolase A or B (Fig.
4).
In this study, the information generated from the primary sequence
alignment of aldolase isozymes is combined with the information generated from the three-dimensional structures of these isozymes to
attribute functional roles for these conserved amino acids. This
analysis revealed two surface patches containing the majority of the
ISRs. These experiments confirm the computational prediction of
isozyme-specific patches distinct from the active site (46). Swapping
the ISRs between two isozymes via site-directed mutagenesis revealed
that the isozyme specificity of aldolase is conferred either through
conformational changes influencing the active site from a distance
and/or causing the C-terminal region to interact directly with the
active site. To explore these possible mechanisms, subsets of ISRs need
to be swapped and subjected to the same analysis as described here to
attribute a role for each conserved residue cluster. It is not likely
that the entire set of ISRs is exclusively involved in influencing the
kinetic differences between isozymes. Specific kinetic roles for
subsets of these ISRs, if any, as well as possible roles in
protein-protein interactions need to be investigated in subsequent studies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
-barrels with the active site of each monomer located at the center of the
/
-barrel. The 18-20 residues at the C
terminus of the protein are likely to be flexible as this portion is
either missing from many structures (10, 12) or found in a variety of
conformations (11, 13). The C-terminal region has been implicated in
determining the isozyme-specific differences of aldolase (14, 15).
Mutations in this region diminish the kinetic distinctions among
isozymes. For example, the swapping of two regions (amino acids 34-55
and the C-terminal region 306-363) of human aldolase A onto human
aldolase B causes changes in the kinetic properties of aldolase B. Like
aldolase A, the chimera had a 5-fold increase in the
kcat and a 4-fold increase in
Km for Fru 1,6-P2 cleavage, as well as a
3-fold decrease in cleavage of Fru 1-P (15). Similarly, the naturally
occurring mutation, A337V, which is located in the C-terminal portion
of human aldolase B, is found in patients with hereditary fructose
intolerance and results in a 43% loss in the ability to cleave Fru 1-P
while maintaining wild-type cleavage rates of Fru 1,6-P2
(16). In contrast, switching the C-terminal 6 residues between isozymes
A and B produces no clear effects on steady-state kinetics (14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helix or
-sheet and those residues
that had side chains accessible to the solvent. In these cases,
replacement with a residue of similar charge and size was unlikely to
alter the overall structure of the protein.
Site-directed mutagenesis
2t = 8.31 × 1011). Circular
dichroism spectra were collected between 185 and 260 nm on an Aviv 62DS
spectrometer equipped with a temperature controller at 20 °C as
described previously (37). Irreversible thermal inactivation was
performed as described previously (37) using 10-min incubations
at temperatures between 30 and 55 °C in 5 °C increments, and the
activity was measured at 30 °C.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Isozyme-specific residues in
vertebrate aldolases. The amino acid sequences of aldolase A
isozymes from various sources were aligned and compared with aldolase B
(17) and C (18) ISRs. Fixed amino acid differences between the
paralogous groups are boxed. The presence of
non-conservative (shaded) and conservative (open)
differences between isozyme groups were evaluated using the
three-dimensional structure of aldolase A (PDB accession code 1ADO)
(13) and the graphics program O (28). Rabbit aldolase A numbering was
used throughout the analysis (57). GenBankTM
accession numbers for the nucleotide sequences are: Human A, M11560;
Mouse A, X61484; Rabbit A, K02300; Rat A, M14420; Chicken A, see
Footnote 2; Xenopus A, AB002267; Human B, K01177; Mouse B,
NM_144903; Rabbit B, U85645; Rat B, M10149; Chicken B, M10946; Sheep B,
Z29372; Sbream B, X82278; Salmon B, AF067796; Human C, X05196; Mouse C,
S72537; Rat C, X06984; Chicken C, S78288; Xenopus C, S73606;
Goldfish C, U36777; Pufferfish C, AF041454. The alignment was generated
using Clustal V (24).
/
-barrel portion of
the protein was chosen. Surprisingly, none of the ISRs were within
hydrogen bonding distance of the active site as defined by the
-amino group of the Schiff base forming Lys-229 and the carboxyl
group of Asp-33 (Fig. 2a). The
closest residue to the active site was 8.2 Å from the catalytic
Asp-33.
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Fig. 2.
The location of ISRs on the tertiary
and quaternary structure of aldolase. The locations of ISRs were
mapped to the tertiary and quaternary structures of aldolase A (PDB
accession code 1ADO) (13) using the program O (28). The ribbon diagram
was created with the programs MOLSCRIPT (58) and POV-Ray (Persistence
of Vision Ray Tracer, version 3.01). The surface renderings were made
with the program GRASP (39). a, ribbon diagram of rabbit
aldolase A looking down the /
-barrel showing the location of ISRs
relative to the active site. ISRs are colored magenta,
green, and yellow for aldolase A, B, and C ISRs,
respectively. Asp-33 and Lys-229 are shown in cyan.
b, the surface model of the aldolase monomer rotated
180o into the plane of the paper relative to panel
a. ISRs are colored as in panel a. The ISR patches are
outlined and labeled.
Steady-state kinetics of wild-type and mutant aldolases
/
-barrel portion of the protein
and were not solvent-accessible. The first ISR surface patch, termed
the distal surface patch (DSP), was related in location to the active
site by a 180o rotation of the monomer. The DSP was
composed of the residues 60, 71, 91, 102, 320, 324, and 332, which were
from different portions of the primary sequence. The second ISR patch
included residues from the C-terminal region (residues 345-353) and
those residues on the surface of the protein proximal to the C-terminal region. This patch was called the TSP and was composed of the residues 38, 39, 40, 45, 311, 314, 350, 353, 354, 358, 359, and 361. The ISRs 350, 353, 354, 358, 359, and 361 were assigned to the TSP
based on the primary sequence since a definitive structure has not been
determined for this region. The remaining surface residues, 113, 156, 262, and 296, were not located in either patch. Excluding the
C-terminal region (residues 345-363), 9 of the ISRs identified here
(38-40, 45, 58, 60, 71, 91, and 102) were located in an area of higher
root-mean-square deviation as compared with the average
root-mean-square deviation of main chain atoms between the aldolase A
and B structures (data not shown). The location of these residues in
areas of structural divergence between the isozymes provided support
for their role in conferring isozyme specificity.
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Fig. 3.
The terminal ISR patch overlaps with an area
of elevated temperature factor. In a, the surface model
of the aldolase A monomer (PDB accession code 1ADO) (13) is shown, and
those residues with an average temperature factor greater than 50 Å2 are colored magenta. In b, the
surface model of the aldolase B monomer (PDB accession code 1QO5) (11)
is shown, and all residues with an average temperature factor greater
than 60 Å2 are colored magenta.
Outlines of the ISR patches, as described in the legend for
Fig. 2, are superimposed. These surface renderings were made
with the program GRASP (39).
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Fig. 4.
The electrostatic potentials mapped onto the
surface of aldolase isozymes. Electrostatic surface potentials of
aldolase A (a), aldolase B (b), and aldolase C
(c) are generated from 1ADO (13), 1QO5 (11), and an aldolase
C homology model, respectively. The model of aldolase C was derived
from the high resolution structure of aldolase A (PDB accession code
1ADO) (13) using the Swiss-Model protein modeling server (38).
Outlines of the ISR patches, as described in the legend for
Fig. 2, are superimposed. Note that the TSP of aldolase B is
different from that of the aldolase A structure. Figures were created
with GRASP (39) and colored based on surface potential in units of k
with the scale (
5.8, 0, 7.3) in GRASP notation where negative values
are red and positive values are blue.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Mary T. Walsh for careful assistance with the circular dichroism experiments.
![]() |
FOOTNOTES |
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* This work was supported by Grant GM60616 from the National Institutes of Health (to D. R. T. and K. N. A.).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.
¶ Present address: The Arabidopsis Information Resource, Department of Plant Biology, Carnegie Institution of Washington, 260 Panama St., Stanford, CA 94305.
Present address: Lawrence Livermore National Laboratory, 7000 East Ave., L-448, Livermore, CA 94551.
** To whom correspondence may be addressed: Dept. of Physiology and Biophysics, Boston University School of Medicine, 715 Albany St., Boston, MA 02118-2394. Tel.: 617-638-4398; Fax: 617-638-4285; E-mail: allen@med-xtal.bu.edu.
To whom correspondence may be addressed: Dept. of Biology,
Boston University, 5 Cummington St., Boston, Massachusetts 02215. Tel.:
617-353-5310; Fax: 617-358-0338; E-mail: tolan@bu.edu.
Published, JBC Papers in Press, February 28, 2003, DOI 10.1074/jbc.M209185200
2 S. Dayanandan and D. R. Tolan, unpublished results.
3 T. Z. Berardini and D. R. Tolan, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: Fru 1, 6-P2, fructose 1,6-bisphosphate; Fru 1-P, fructose 1-phosphate; ISR, isozyme-specific residue; TSP, terminal surface patch; DSP, distal surface patch; PDB, Protein Data Bank.
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