Besides the R67S and R91T mutant enzymes, which are insensitive towards fumarate, the E59L mutant enzyme does not show any enzyme activation with fumarate, indicating that Glu59 has rema
Trang 1allosteric regulation and subunit–subunit interaction of
Ju-Yi Hsieh1,*, Yu-Hsiu Chiang1,*, Kuan-Yu Chang1and Hui-Chih Hung1,2
1 Department of Life Sciences, National Chung-Hsing University, Taichung, Taiwan
2 Institute of Bioinformatics, National Chung-Hsing University, Taichung, Taiwan
Malic enzyme (ME) comprises a family of oxidative
decarboxylases that catalyze the transformation of the
substrate l-malate to CO2 and pyruvate, with
instanta-neous reduction of NAD(P)+ to NAD(P)H [1–3]
Divalent metal ions (Mn2+or Mg2+) are essential for
this enzymatic reaction These enzymes are universally present in nature, with conserved sequences, and have generally similar structural topology among different species [4–8] According to their cofactor specificity, mammalian ME has been divided into three isoforms:
Keywords
allosteric regulation; analytical
ultracentrifugation; electrostatic interaction;
malic enzyme; mutagenesis
Correspondence
H.-C Hung, Department of Life Sciences
and Institute of Bioinformatics, National
Chung-Hsing University, 250, Kuo-Kuang
Road, Taichung, 40227 Taiwan
Fax: +886 4 22851856
Tel: +886 4 22840416 (ext 615)
E-mail: hchung@dragon.nchu.edu.tw
*These authors contributed equally to this
work
(Received 30 July 2008, revised
19 November 2008, accepted 4
December 2008)
doi:10.1111/j.1742-4658.2008.06834.x
Here we report on the role of Glu59 in the fumarate-mediated allosteric regulation of the human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) In the present study, Glu59 was substituted by Asp, Gln or Leu Our kinetic data strongly indicated that the charge properties of this residue significantly affect the allosteric activation of the enzyme The E59L enzyme shows nonallosteric kinetics and the E59Q enzyme displays a much higher threshold in enzyme activation with elevated activation con-stants, KA,Fum and aKA,Fum The E59D enzyme, although retaining the allosteric property, is quite different from the wild-type in enzyme activa-tion The KA,Fumand aKA,Fum of E59D are also much greater than those
of the wild-type, indicating that not only the negative charge of this residue but also the group specificity and side chain interactions are important for fumarate binding Analytical ultracentrifugation analysis shows that both the wild-type and E59Q enzymes exist as a dimer–tetramer equilibrium In contrast to the E59Q mutant, the E59D mutant displays predominantly a dimer form, indicating that the quaternary stability in the dimer interface
is changed by shortening one carbon side chain of Glu59 to Asp59 The E59L enzyme also shows a dimer–tetramer model similar to that of the wild-type, but it displays more dimers as well as monomers and polymers Malate cooperativity is not significantly notable in the E59 mutant enzymes, suggesting that the cooperativity might be related to the molecu-lar geometry of the fumarate-binding site Glu59 can precisely maintain the geometric specificity for the substrate cooperativity According to the sequence alignment analysis and our experimental data, we suggest that charge effect and geometric specificity are both critical factors in enzyme regulation Glu59 discriminates human m-NAD-ME from mitochondrial NADP+-dependent malic enzyme and cytosolic NADP+-dependent malic enzyme in fumarate activation and malate cooperativity
Abbreviations
c-NADP-ME, cytosolic NADP+-dependent malic enzyme; ES, enzyme–substrate; ME, malic enzyme; m-NAD-ME, mitochondrial NAD(P)+ -dependent malic enzyme; m-NADP-ME, mitochondrial NADP + -dependent malic enzyme; PFK-1, phosphofructokinase-1.
Trang 2cytosolic NADP+-dependent ME (c-NADP-ME)
[9,10], mitochondrial NADP+-dependent ME
(m-NADP-ME) [11], and mitochondrial NAD(P)+-dependent ME
(m-NAD-ME) [1,11] Mitochondrial NAD(P)+
-depen-dent ME has dual cofactor specificity, and can use
both NAD+and NADP+as cofactor, but
physiologi-cally it favors NAD+in maximizing its enzyme
activ-ity [1,12] Human m-NAD-ME may associated with
the growth of highly proliferating tissues and tumors
through the NADH and pyruvate produced in
gluta-minolysis [1,13–20]
Unlike the other two mammalian isoforms,
m-NAD-ME is a regulatory enzyme with a complex
control system for manipulating its catalytic activity
[21–23] The enzyme exhibits positive cooperative
behavior with respect to the substrate l-malate, and it
is an allosteric enzyme activated by fumarate [18,21–
27] Previous studies have suggested that ATP may act
as an allosteric inhibitor of m-NAD-ME [18,21,25],
and the allosteric properties of this isoform may relate
to its particular role in the pathways of malate and
glutamine oxidation in tumor mitochondria [17–21,24]
However, further site-directed mutagenesis and kinetics
studies showed that ATP may actually act as an active
site inhibitor, rather than an allosteric inhibitor
[28,29]
The crystal structures of MEs demonstrate that the
enzyme is a homotetramer with a double-dimer
qua-ternary structure On the basis of structural
informa-tion, MEs are categorized into a new class of
oxidative decarboxylases with a novel backbone
struc-ture [4,6,8,30] The strucstruc-tures of human m-NAD-ME
with malate⁄ pyruvate, Mn2+⁄ Mg2+, NAD+,
fuma-rate and transition state analog inhibitors have been
resolved [4,5,31–33] In the structure of human
m-NAD-ME, besides the active site, there are two
regulatory sites (Fig 1A) One of them, located at the
tetramer interface and called the exo site, is occupied
by an NAD or ATP molecule The other is at the
dimer interface, and is occupied by fumarate [32] The
structures of pigeon c-NADP-ME and Ascaris suum
m-NAD-ME in complex with various ligands have
also been reported [6,7,30] These structures do not
show the additional exo site, but a separate allosteric
site is found in the A suum m-NAD-ME at the dimer
interface [26,27] Figure 1B shows the binding mode
of fumarate at the dimer interface Structural studies
have revealed that Arg67 and Arg91 are the ligands
for fumarate binding The side chains of Arg91 and
Arg67 form salt bridges with the carboxylate group of
fumarate (Fig 1B) Site-directed mutagenesis and
kinetic studies confirmed that Arg67 and Arg91 are
indeed essential for fumarate activation Both R67S
and R91T mutant enzymes are insensitive towards fumarate [32] However, both Arg67 and Arg91 are conserved among other ME isoforms that are not activated by fumarate (Fig 1C) Thus, additional fac-tors must be involved in governing the activation mechanism of fumarate in the human and A suum m-NAD-ME [27,32]
In our previous work, we delineated the functional role of Asp102, which is close to the Arg67–fumarate– Arg91 ion pair network but does not directly interact with fumarate and is not conserved in other nonallos-teric MEs (Fig 1C) We proposed that Asp102 is important for preserving the electrostatic balance in the fumarate-binding pocket, which may be a central factor in the regulatory mechanism of fumarate Muta-tion of Asp102 to Ala and Lys, however, abolishes the allosteric activation of the enzyme [23]
In this article, we aimed to explore in detail the fac-tors governing the allosteric regulation of the enzyme Previous studies have already shown that Glu59, a structural neighbor of Arg67, plays an important role
in the fumarate activation of the enzyme Besides the R67S and R91T mutant enzymes, which are insensitive towards fumarate, the E59L mutant enzyme does not show any enzyme activation with fumarate, indicating that Glu59 has remarkable effects on allosteric activa-tion [32] In the present study, the funcactiva-tional roles of Glu59 in the regulatory mechanism of the enzyme are elucidated Glu59 is substituted by Asp, Gln and Leu Detailed kinetic and analytical ultracentrifugation analyses of these mutants help to determine the factors affecting fumarate activation, subunit–subunit interaction and substrate cooperativity of human m-NAD-ME
Results
Kinetic parameters of the human wild-type and E59 mutant m-NAD-MEs
The kinetic parameters of the wild-type and E59 mutant enzymes were determined with and without
20 mm fumarate (Table 1) Without fumarate, there were no significant differences in Km,NAD and
K0.5,Malate observed among the wild-type and E59 mutant enzymes, except for the E59D enzyme The
Km,NADand K0.5,Malatevalues of E59D were 2-fold and 4.4-fold higher, respectively, than that of the wild-type Furthermore, the kcat value of E59 mutant enzymes was only one-third to one-quarter of that of the wild-type, suggesting that mutation of Glu59 in the fumarate-binding site causes the enzyme to become less efficient in catalysis
Trang 3C
D
B
Fig 1 binding site for human m-NAD-ME (A) Homotetramer of human m-NAD-ME (Proein Data Bank code: 1PJ3) (B) Fumarate-binding site of m-NAD-ME The corresponding amino acids in the fumarate-Fumarate-binding site, Arg67, Arg91 and Glu59, are represented as a ball-and-stick model The color is yellow for fumarate This figure was generated with PYMOL (DeLano Scientific LLC, San Carlos, CA, USA) (C) Multiple sequence alignments of three clusters of ME isoforms around the fumarate-binding region are shown Amino acid sequences of MEs were obtained by a similarity search of BLAST [44], and alignments were created with CLUSTAL W [45] This figure was generated using the BIOEDIT sequence alignment editor program [46] (D) Glu59-binding ligands in the wild-type enzyme are shown as the LIGPLOT diagram [47] The bold bonds indicate the specific amino acid, the thin bonds are the hydrogen-bonded residues, and the green dashed lines corre-spond to the hydrogen bonds Spoked arcs represent hydrophobic contacts.
Trang 4With fumarate, the Km,NAD and K0.5,Malate of the
wild-type enzyme decreased, the kcat values of the
enzyme increased, and the h value, which represents
the cooperativity of malate binding, was significantly
reduced from 1.8 to 1, indicating the characteristics of
allosteric regulation of the enzyme isoform by
fuma-rate The E59Q enzyme, similar to the wild-type,
showed a decrease in Km,NAD and K0.5,Malate but an
increase in kcat However, a considerable decrease in
malate cooperativity was observed in this mutant The
h value was 1.2 with or without fumarate For the
E59D enzyme, the Km,NAD and K0.5,Malate values
decreased by about two-fold and 10-fold, respectively,
and the kcat value of the mutant enzyme increased by
three-fold The malate cooperativity, however,
com-pletely disappeared in this mutant The h value was
1.0, with or without fumarate Indeed, the E59D
enzyme had an unusually large K0.5,Malatevalue, which
could be reduced to a level similar to that of the
wild-type by the addition of fumarate, suggesting that the
active site of this mutant had been changed and was
readjusted by fumarate For the E59L enzyme, the
K0.5,Malate and kcatvalues were not notably influenced
by fumarate, indicating that these mutant enzymes
were insensitive to fumarate activation
Activating effect of fumarate on the human
wild-type and E59 mutant m-NAD-MEs
The initial rates of m-NAD-ME measured in various
concentrations of fumarate showed hyperbolic kinetics
(Fig 2) At a saturating concentration of fumarate, the
maximal activation by fumarate for the wild-type
enzyme was approximately 1.5-fold, with an apparent
KA value of 0.21 ± 0.03 mm (Fig 2, closed circles)
The maximal activation fold of the E59Q enzyme was similar to that of the wild-type enzyme, whereas the relative enzyme activity of the E59Q enzyme was only one-third of that of the wild-type (Fig 2, closed trian-gles), and it had a much higher apparent KA value (14.1 ± 2.3 mm) than that of the wild-type, suggesting that the E59Q enzyme needed more fumarate mole-cules to achieve its maximal activation The E59D enzyme could be activated more than three-fold, with
an apparent KA value of 6.1 ± 1.1 mm, a value that was also much higher than that of the wild-type (Fig 2, open circles) Even though the maximal activa-tion fold of the E59D enzyme was more than that of the wild-type, the relative enzyme activity of the E59D enzyme was raised to the level of one-half of that of the wild-type (Fig 2, open circles) Like the E59Q enzyme, the E59D enzyme also required more fuma-rate to achieve this activation The E59L enzyme could not be activated by fumarate (Fig 2, open triangles) These preliminary results indicate that the side chain properties of residue 59 seem to have a great impact
on the allosteric regulation of ME
Activation constants of the human wild-type and E59 mutant m-NAD-MEs
The activation constants of the wild-type and E59 mutant enzymes were further determined by kinetic analysis The enzyme activities were assayed in a broad range of substrate malate concentrations at different fixed concentrations of fumarate (data not shown) These curves were globally fitted to Eqn (1), and the activation constant of fumarate for free enzyme (KA,Fum) and for the enzyme–substrate (ES) complex (aKA,Fum) were estimated
Table 1 Kinetic parameters for the human wild-type and E59
mutant m-NAD-MEs ), no fumarate added; +, with 20 m M
fuma-rate added.
Km,NAD
(m M )
K0.5,Malate (m M ) kcat(s)1) h Wild-type
) 0.88 ± 0.10 15.44 ± 0.97 208 ± 10.6 1.84 ± 0.21
+ 0.43 ± 0.04 3.28 ± 0.37 332 ± 15.3 1.01 ± 0.09
E59Q
) 0.92 ± 0.10 15.23 ± 2.10 74 ± 7.5 1.24 ± 0.13
+ 0.82 ± 0.06 7.01 ± 1.13 141 ± 10.3 1.21 ± 0.07
E59D
) 1.60 ± 0.16 67.22 ± 7.76 57 ± 3.5 1.03 ± 0.05
+ 0.76 ± 0.04 6.43 ± 0.68 196 ± 8.6 1.01 ± 0.11
E59L
) 0.76 ± 0.06 10.68 ± 1.03 53 ± 3.1 1.20 ± 0.14
+ 0.49 ± 0.04 10.75 ± 1.91 50 ± 4.1 1.00 ± 0.08
[Fumarate] (mM)
0 20 40 60 80 100
Fig 2 Fumarate activation of the human wild-type and E59 mutant m-NAD-MEs The assay mixture contained ME (1.5 lg), 40 m M
malate, 10 m M MgCl 2 , and 2 m M NAD + , with various fumarate concentrations as indicated Closed circles, wild-type enzyme; closed triangles, E59Q enzyme; open circles, E59D enzyme; open triangles, E59L enzyme.
Trang 5The activation parameters of the wild-type and E59
mutant human enzymes are summarized in Table 2
The values of KA,Fum and aKA,Fum with respect to
malate for the wild-type enzyme were 0.73 mm and
0.17 mm, respectively, indicating that fumarate binds
the ES complex more tightly than free enzyme For the
E59Q enzyme, the values of KA,Fumand aKA,Fumwere
13.1 and 5.63 mm, respectively, greater than those of
the wild-type by at least an order of magnitude
(18-fold and 33-(18-fold, respectively), indicating that the
bind-ing affinity of fumarate for the E59Q enzyme was
markedly less than that of the wild-type The negative
charge of Glu59 is important for the binding affinity
of fumarate to either free enzyme or the ES complex
For the E59D enzyme, the KA,Fumand aKA,Fumvalues
were 8.5 and 3.91 mm, respectively, also larger than
those of the wild-type enzyme by over 10-fold This
reveals that although the negative charge was
con-served, the activation constants of fumarate for the
E59D enzyme were considerably elevated, reflecting a
decreased binding affinity of fumarate
Self-association of the human wild-type and E59
mutant m-NAD-MEs
The fumarate-binding site resides at the dimer interface
(Fig 1A) We use analytical ultracentrifugation to
examine the possible change in the quaternary
struc-ture of E59 mutant enzymes Figure 3 shows the
con-tinuous sedimentation coefficient distribution of the
wild-type and E59 mutants The sedimentation
coeffi-cients of 6.5 S and 9.0 S represented the dimer and
tetramer, respectively, corresponding to molecular
masses of 124 and 248 kDa The quaternary structure
of the wild-type is in dimer–tetramer equilibrium with
different protein concentrations (Fig 3A–C) The
E59Q enzyme apparently shows a similar dimer–
tetramer pattern as the wild-type (Fig 3D–F), both
being in dimer form in low protein concentrations and
being reconstituted into tetramers in high protein
concentrations In contrast to the E59Q enzyme, the
E59D enzyme has a predominantly dimer form, with a
few monomers and polymers in the range of protein concentrations used (Fig 3G–I), indicating that the quaternary stability in the dimer interface is greatly changed by shortening one carbon side chain of Glu59
to Asp59 The E59L enzyme also showed a dimer– tetramer model similar to that of the wild-type, but it displayed more dimers as well as monomers and poly-mers, which are not observed to a significant extent in the wild-type WT (Fig 3J–L)
In order to estimate the self-association of enzymes quantitatively, the sedimentation velocity data were analyzed globally to determine the dissociation constant (Kd) of the wild-type and E59 mutants (Fig 4) Struc-tural data show that the interactions in the tetramer interface are weaker than those in the dimer interface (Fig 1A); thus, the Kd value of the wild-type may reflect the dissociation between the A and D or B and C subunits to form AB or CD dimers The wild-type and E59Q enzymes had similar Kdvalues of 6.3 and 7.9 lm, respectively (Table 2), showing that the subunit–subunit interactions in the dimer interface were not disrupted
by substituting a negatively charged Glu with a neutrally charged Gln The E59D enzyme, although conserving the negative charge on residue 59, still altered its dimer–tetramer equilibrium into a dominant dimer form with a Kdvalue of 4395 lm, which is over 600-fold larger than that of the wild-type As Glu59 is near the dimer interface, the E59D dimer might be an
AD (BC) dimer If this is the case, the Kdvalue of the E59D enzyme may represent the dissociation in the dimer interface The E59L enzyme is also in dimer– tetramer equilibrium, with a slightly larger Kd value than the wild-type, suggesting that the Leu substitution did not cause substantial changes in the dimer interface
In the presence of fumarate, the dimer–tetramer equilibrium of the wild-type was shifted (Fig 5A) With the addition of fumarate, most dimeric enzymes reconstitute into tetrameric enzymes, suggesting that fumarate stabilizes the tetrameric state of the enzyme, which may increase the catalytic activity Similar to the wild-type, the E59Q enzyme was also changed in its dimer–tetramer equilibrium by fumarate (Fig 5B), suggesting that the tetramer organization was not perturbed in this mutant The E59D enzyme, however, could not be reconstituted into a tetramer by fumarate (Fig 5C); it existed in a dimeric form in the presence
of fumarate Although the quaternary structure of the E59L enzyme displayed a model similar to that of the wild-type (Fig 3), the dimer–tetramer equilibrium of this mutant enzyme could not be shifted by fumarate (Fig 5D), suggesting that the mutant enzyme is insen-sitive to fumarate with regard not only to catalytic activity but also to quaternary structure organization
Table 2 Activation constants of fumarate and dissociation
con-stants of dimer–tetramer equilibrium for the human wild-type and
E59 mutant m-NAD-MEs.
KA,Fum(m M )
aKA,Fum (m M ) Kd(l M )
DG (kcalÆmol)1) Wild-type 0.73 ± 0.11 0.17 ± 0.03 6.27 ± 0.05 )7.0 ± 0.05
E59Q 13.1 ± 3.9 5.63 ± 1.68 7.93 ± 0.06 )6.8 ± 0.05
E59D 8.5 ± 1.8 3.91 ± 0.83 4395 ± 23 )3.2 ± 0.02
Trang 6Binding network of the allosteric activator
fumarate in human m-NAD-ME
Fumarate has been identified as the allosteric activator
for human m-NAD-ME by decreasing the Km values
of the active site ligands [13,21,23,28] As well as the
mammal enzymes, m-NAD-ME from A suum is also activated by fumarate [27] As the crystal structure of human m-NAD-ME in complex with fumarate has been determined, the binding network of fumarate in the enzyme has become clear In the fumarate-binding pocket, two arginyl residues, Arg67 and Arg91, have been identified as determining the major binding affin-ity of fumarate for the enzyme An anionic amino
Fig 3 Continuous sedimentation coefficient distribution of the human wild-type and E59 mutant m-NAD-MEs The enzymes were used at three protein concentrations, 0.2, 0.6 and 1.2 mgÆmL)1in 50 m M Tris ⁄ HCl buffer (pH 7.4) at 20 C (A–C) Wild-type (D–F) E59Q (G–I) E59D (J–L) E59L.
Trang 7acid, Glu59, which forms salt bridges with Arg67 in
the structure but is not found in nonallosteric MEs, is
suggested to be important for fumarate activation [32]
In this study, we examined the effect of the charge and
hydrogen bonding network of the Glu59 side chain on
allosteric regulation of the enzyme
Charge effect of residue 59 on enzyme regulation
In the structure of the wild-type enzyme, Glu59 is ion-paired and hydrogen-bonded with Arg67 and Lys57 (Fig 1D) Simultaneously, fumarate is coordinated with Arg67 and Arg91 in the structure; the ionic pairs
Fig 4 Global analysis of sedimentation velocity data of the human wild-type and E59 mutant m-NAD-MEs at three protein concentrations Sedimentation was performed at 20 C with an An-50 Ti rotor and at a rotor speed of 42 000 r.p.m (A–C) Concentrations of the protein were 0.2, 0.6 and 1.2 mgÆmL)1, respectively The symbols are raw sedimentation data, and the lines are data fitted by the software SEDPHAT (D–F) The fitting residuals of the model from the upper panel afford a reliable analysis result for a dissociation constant (Kd) of the mono-mer–dimer equilibrium.
Fig 5 Continuous sedimentation coefficient distribution of the human wild-type and E59 mutant m-NAD-MEs in the presence of fumarate The enzyme concentration was fixed at 0.2 mgÆmL)1without (solid lines) or with (broken lines) 1 m M (broken lines) or 3 m M (dotted lines) fumarate (A) Wild-type (B) E59Q (C) E59D (D) E59L.
Trang 8and hydrogen bonding network among Glu59, Arg67,
fumarate and Arg91 may be responsible for
fumarate-induced allosteric regulation (Fig 1B) Our kinetic
data strongly indicated that the charge properties of
Glu59 significantly affect the allosteric activation of
the enzyme (Fig 2) The fact that the E59L enzyme
shows nonallosteric kinetics and the E59Q enzyme
dis-plays a much higher threshold for enzyme activation
clearly indicates that the charge effect is an influential
factor in fumarate activation Abolishing the salt
bridges between Glu59 and Arg67 has a significant
effect on the binding network of fumarate
The E59Q enzyme can still be activated by fumarate,
and the maximal activation fold is similar to that of
the wild-type, whereas achieving the maximal
activa-tion requires a higher fumarate concentraactiva-tion
Signifi-cant differences in KA,Fum and aKA,Fum were observed
for the E59Q enzyme, suggesting that the negative
charge of Glu has a significant impact on the binding
affinity of fumarate As the molecular dimensions and
polarity of Gln are close to those of Glu, the proper
conformational geometry and hydrogen bonding
net-work should be preserved even though the negative
charge of Glu is replaced by a neutral side chain of
Gln Thus, the delayed activation of this mutant
enzyme could be attributed to the ion pairs derived
from Glu59 being destroyed by this replacement
For the E59D enzyme, despite the negative charge
of the residue and the allosteric property of the
enzyme being retained, the kinetic properties are quite
different from those of the wild-type The KA,Fum and
aKA,Fum values of the E59D enzyme are also much
greater than those of the wild-type, indicating that not
only the negative charge of this residue but also the
group specificity and side chain interactions are
impor-tant for fumarate binding Furthermore, kinetic
analy-sis demonstrates that the E59D enzyme resembles a
partial inactive enzyme with an anomalously high Km
value and low enzyme activity in the absence of
fuma-rate This may result from shortening the length of the
side chain in the enzyme, causing geometrical changes
in the fumarate-binding pocket, which may have an
effect on the active site The structural change in the
E59D enzyme may be adjusted by binding of fumarate,
and the mutant enzyme can thus be reactivated
Subunit–subunit interaction in the dimer
interface and malate cooperativity
Analytical ultracentrifugation analysis demonstrates
that the wild-type enzyme exists in dimer–tetramer
equilibrium in solution The quaternary structure of
the E59Q enzyme is as stable as that of the wild-type,
demonstrating a similar dimer–tetramer pattern (Figs 3 and 5) and a similar dissociation constant (Table 2) This fact suggests that the molecular geometry in the dimer interface is not significantly changed by the substitution of Gln
Unlike the E59Q enzyme, the E59D enzyme exists mainly as a dimer rather than in dimer–tetramer equilib-rium (Fig 3) The dissociation of subunits in the dimer interface might be caused by the structural change in the E59D enzyme The ionic interactions between Asp59 and Lys57 might still occur, but be somewhat altered because of the shorter side chain of Asp59 In the wild-type, Lys57 is located in the dimer interface; its side chain is hydrogen-bonded not only with Glu59 but also with Pro216 and Tyr218 from the other subunit Analyt-ical ultracentrifugation analysis shows that mutation of Lys57 causes the enzyme to dissociate into unstable dimers and, further, to form polymers (J.-Y Hsieh,
Y.-W Fang & H.-C Hung, unpublished results) In the E59D enzyme, Lys57 might be pulled by Asp59 into one subunit, thus being no longer hydrogen-bonded with Pro216 and Tyr218 from the other subunit, and finally leading to the disintegration of tetramers of the enzyme This can be fitted well with the explanation of why the E59L enzyme is kept in dimer–tetramer equilibrium It can be concluded that the hydrophobic Leu introduced did not influence the subunit interactions of Lys57 However, the monomer and polymer appearing in the equilibrium may have been caused by the environmental alteration from hydrophilic to hydrophobic
Although Glu59 is near the dimer interface, it is also possible that the E59D dimers are AB (and CD) dimers, based on the fact that the tetramer is a dimer
of AB (and CD) dimers Another possibility could be that the E59D enzyme maintains a disrupted AB dimer, and the conformational disturbance somehow prevents the formation of the tetramer However, this question can be definitively answered until the biophysical data for discrimination between AB and
AD dimers are available
The malate cooperativity in the E59 mutant enzymes
is almost abolished This is not surprising in the E59D enzyme, because this mutant enzyme is mainly in dimer form For the E59Q and E59L enzymes, even though the former conserves the allosteric activation of fumarate, and like the wild-type, both principally reserve a dimer–tetramer equilibrium, their malate cooperativity still decreases significantly The loss of cooperativity might be related to the alteration in molecular geometry of the fumarate-binding site Our data suggest that only Glu59 can precisely hold the geometric specificity of the allosteric site and that this
is important for substrate cooperativity
Trang 9Involvement of the allosteric triad in the
regulatory mechanism of human m-NAD-ME
Structural data have clearly revealed that both Arg67
and Arg91 are the direct ligands for fumarate It is not
surprising that mutation of these argininyl residues
causes the enzyme to become insensitive to fumarate
[32] According to the sequence alignments, neither
Arg67 nor Arg91 are specifically conserved in m-NAD
ME, the only isoform that could be activated by
fuma-rate In fact, Arg91 is highly conserved among all
clas-ses of MEs, whereas Arg67, although moderately
conserved, is completely conserved among all
mam-malian ME isoforms (Fig 1E) Glu59, however, is
conserved only in m-NAD-ME (Fig 1C); in
m-NADP-ME and c-NADP-m-NADP-ME, this residue is replaced by Leu
or Asn, respectively Hence, the latter two enzymes are
not fumarate-activated In fact, the E59L and E59Q
enzymes display, as such, the properties of a
nonregu-latory ME The E59L m-NAD-ME is insensitive to
fumarate, showing nonallosteric and noncooperative
kinetics, whereas E59Q m-NAD-ME displays a much
higher activation threshold and less cooperativity
Glu59 thus discriminates human m-NAD-ME from
m-NADP-ME and c-NADP-ME in fumarate
regula-tion and malate cooperativity Glu59, Arg67 and
Arg91 form an allosteric triad in the fumarate site of
human m-NAD-ME The allosteric triad is the basic
element for fumarate binding, and thus determines
whether the enzyme is allosteric or nonallosteric
Many enzymes in the metabolic pathway are
controlled by its allosteric regulator
Besides human m-NAD-ME, many enzymes in the
metabolic pathway are allosteric enzymes The one
that is most well characterized is
phosphofructokinase-1 (PFK-phosphofructokinase-1) [34] PFK-phosphofructokinase-1 catalyzes the phosphorylation
of fructose 6-phosphate to fructose 1,6-bisphosphate
Fructose 2,6-bisphosphate is the potent allosteric
acti-vator for this regulatory enzyme [35,36] In the absence
of fructose 2,6-bisphosphate, the enzyme is almost
inactive at the physiological concentrations of its
sub-strate, fructose 6-phosphate When fructose
2,6-bis-phosphate binds to the allosteric site on PFK-1, it
increases the substrate affinity, with a significant
decrease of K0.5from 2 mm to 0.08 mm Similar to the
effect of fumarate on human m-NAD-ME, fructose
2,6-bisphosphate activates PFK-1 by increasing the
apparent affinity for fructose 6-phosphate The
regula-tory mechanism of these two enzymes gives a good
example of the reactivation–deactivation of an
alloste-ric enzyme controlled by its specific allostealloste-ric regulator produced in the metabolic pathway
Experimental procedures
Expression and purification of recombinant MEs The detailed expression and purification protocols for human m-NAD-ME have been reported in earlier studies [1,31] In brief, m-NAD-ME was subcloned into the expres-sion vector (pRH281) and transformed into Escherichia coli BL21 cells for enzyme overexpression by controlling the inducible trp promoter system [1] Anionic exchange, DEAE–Sepharose (Amersham Biosciences, Uppsala, Swe-den), followed by ATP–agarose affinity chromatography (Sigma, St Louis, MO, USA) were employed in the enzyme purification The purified enzyme was subsequently buffer-exchanged and concentrated in 30 mm Tris⁄ HCl (pH 7.4) and 2 mm b-mercaptoethanol by a centrifugal filter device (Amicon Ultra-15; Millipore, Billerica, MA, USA) with a molecular mass cutoff of 30 kDa The enzyme purity was checked by SDS⁄ PAGE, and the protein concentrations were estimated by the Bradford method [37]
Site-directed mutagenesis Site-directed mutagenesis was carried out using the Quik-Change kit (Stratagene, La Jolla, CA, USA) The purified DNA of human m-NAD-ME was used as a template, and the primers with the desired codons were employed to change Glu59 into Asp, Gln and Leu, using a high fidelity of Pfu DNA polymerase in the PCR reaction Primers including the mutation site are 25- to 45-mer, which is considered nec-essary for specific binding of template DNA The synthetic oligonucleotides used in these site-directed mutagene-sis experiments were 5¢-GGACTTCTACCTCCCAAAATA GACACACAAGATATTCAAGCC-3¢ for E59D, 5¢-GGAC TTCTACCTCCCAAAATACAGACACAAGATATTCAA GCC-3¢ for E59Q, and 5¢-GGACTTCTACCTCCCAAAA TACTGACACAAGATATTCAAGCC-3¢ for E59L The nucleotides underlined and marked in bold indicate the mutation positions After 16–18 temperature cycles, the mutated plasmids including staggered nicks were made The PCR products were subsequently treated with DpnI to digest the wild-type human m-NAD-ME templates Finally, the nicked DNA with desired mutations was trans-formed into E coli strain XL-1, and their DNA sequences were checked by autosequencing
Enzyme kinetic analysis Enzymatic activity of MEs was measured by the reduction
of NAD+ to NADH The reaction mixture contained
Trang 1050 mm Tris⁄ HCl (pH 7.4), 40 mm malate (pH 7.4), 2.0 mm
NAD+and 10 mm MgCl2in a total volume of 1 mL The
absorbance at 340 nm at 30C was instantaneously traced
after the enzyme was added to the reaction mixture, and
monitored continuously in a Beckman DU 7500
spectro-photometer Under these conditions, 1 unit of the enzyme
was defined as the amount of enzyme catalyzing the
produc-tion of 1 lmol of NADH per min An extincproduc-tion coefficient
of 6.22 per mm for NADH was utilized in the calculations
Apparent Michaelis constants of the substrate and cofactors
were determined by varying the concentration of one
sub-strate (or cofactor) around its Kmvalue, while keeping other
components constant at the saturating concentrations The
nonessential activation model was employed to estimate the
dissociation constants for free enzyme (E) and ES complex
[38] The experiment was carried out at a series of fumarate
concentrations and at different concentrations of l-malate
The total set of data was globally fitted to the following
equation, which was derived from a nonessential activation
mechanism (Scheme 1) [38]:
m=Vmax¼ ½S=fKs ð1 þ ½A=KA;FumÞ=ð1 þ b½A=aKA;FumÞ
þ ½S ð1 þ ½A=KA;FumÞ=ð1 þ b½A=aKA;FumÞg ð1Þ
in which v is the observed initial velocity, and Vmax is the
maximum rate of the unactivated reaction The maximum
rate in the presence of fumarate is bVmax Ksis the
Micha-elis constant for the substrate, and KA,Fumand aKA,Fumare
the activation constants for fumarate binding to free
enzyme (E) and ES complex, respectively
The sigmoidal curves of [malate] versus initial rates
were fitted into the Hill equation, and data were further
analyzed to calculate the K0.5 value, the substrate
con-centration at half-maximal velocity, and the Hill
coeffi-cient (h), which were employed to assess the degree of
cooperativity
m¼ Vmax½malateh=ðKh
0:5þ ½malatehÞ All datafitting work was carried out with the sigma
-plot8.0 program (Jandel, San Rafael, CA, USA)
Quaternary structure analysis by analytical ultracentrifugation
Sedimentation velocity experiments were carried out using
a Beckman Optima XL-A analytical ultracentrifuge Sample (380 lL) and buffer (400 lL) solutions were loaded into the double sector centerpiece separately, and built up in a Beckman An-50 Ti rotor Experiments were performed at
20C and a rotor speed of 42 000 r.p.m Protein samples were monitored by UV absorbance at 280 nm in a continu-ous mode with a time interval of 480 s and a step size of 0.002 cm Multiple scans at different time points were fitted
to a continuous size distribution model by the program sedfit[39–42] All size distributions were solved at a confi-dence level of P = 0.95, a best-fitted average anhydrous frictional ratio (f⁄ f0), and a resolution N of 200 sedimenta-tion coefficients between 0.1 and 20.0 S
To precisely determine the dissociation constants of MEs
in dimer–tetramer equilibrium, sedimentation velocity experiments were performed with three different protein concentrations of the enzyme All sedimentation data were globally fitted to the monomer–dimer equilibrium model of the program sedphat to calculate the dissociation constant (Kd) of the enzyme [41] The partial specific volume of the enzyme, solvent density and viscosity were calculated by the software program sednterp [43]
Acknowledgements
This work was supported by the National Science Council, ROC (NSC-96-2311-B-005-005 to H.-C Hung), and in part by the Ministry of Education, Tai-wan, ROC under the ATU plan We thank Professor
G G Chang (Faculty of Life Sciences and the Insti-tute of Biochemistry, National Yang-Ming University) for critically reading the manuscript
References
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KA,Fum
Ks
ES
KA,Fum
Scheme 1 Nonessential activation mechanism of human
mito-chondrial NAD(P) + -dependent malic enzyme.