Báo cáo khoa học: Critical roles of conserved carboxylic acid residues in pigeon cytosolic NADP+-dependent malic enzyme docx

pigeon cytosolic NADP+-dependent malic enzymeShuo-Chin Chang1*, Kuan-Yu Lin1*, Yu-Jung Chen1, Chin-Hung Lai1, Gu-Gang Chang2 and Wei-Yuan Chou1 1 Department of Biochemistry, National Def

pigeon cytosolic NADP+-dependent malic enzyme

Shuo-Chin Chang1*, Kuan-Yu Lin1*, Yu-Jung Chen1, Chin-Hung Lai1, Gu-Gang Chang2

and Wei-Yuan Chou1

1 Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan

2 Faculty of Life Sciences, Institute of Biochemistry, Structural Biology Program, National Yang-Ming University, Taipei, Taiwan

Cytosolic NADP+-dependent malic enzyme (EC

1.1.1.40) catalyses the decarboxylation of l-malate to

pyruvate with oxaloacetate as intermediate and is

asso-ciated with the reduction of NADP+ to NADPH in

the presence of a bivalent metal ion Malic enzyme is a

tetramer of identical subunits The 3D structures of

malic enzymes have been studied extensively [1] Since

the first crystal structure was solved for the human

mitochondrial NAD(P)+-dependent malic enzyme

complexed with Mn2+and ATP [2], 14 structures from

various species, including the human, pigeon, the

roundworm Ascaris suum, and the bacterium

Thermo-toga maritima, have been deposited in the protein

data-bank All these structures, except that of T maritima,

have similar topology These structures are classified

into open and closed forms, depending on the presence

of the substrate, l-malate, or its analogues [3] It has been proposed that the closed form is the catalytically active form of the enzyme

Based on pH profiles and isotope studies of malic enzyme, it was proposed that its catalysis involves a general acid⁄ base mechanism [4–7] A general base is involved in deprotonating the C2 hydroxy group to form an oxaloacetate intermediate and in facilitating the hydride transfer from C2 to NADP+ After decarboxylation of oxaloacetate, a general acid partici-pates in the enol–keto tautomerization of pyruvate Site-directed mutagenesis and kinetic results suggest that K199 in Ascaris (K162 in pigeon) [8] and D295 (D258 in pigeon) [9] function as the general acid and base, respectively Our previous studies indicated that the K162 residue of pigeon NADP+-dependent malic

Keywords

chemical rescue; general acid ⁄ base; malic

enzyme; metal ion binding; site-directed

mutagenesis

Correspondence

W.-Y Chou, Department of Biochemistry,

National Defense Medical Center,

161 MinQuan E Road Sec 6, Taipei,

Taiwan 11490

Fax: +886 2 8792 3106

Tel: +886 2 8791 0776

E-mail: wyc@mail.ndmctsgh.edu.tw

*These authors contributed equally to the

experimental work.

(Received 19 May 2006, revised 2 July

2006, accepted 7 July 2006)

doi:10.1111/j.1742-4658.2006.05409.x

Malic enzyme catalyses the reduction of NADP+ to NADPH and the decarboxylation of l-malate to pyruvate through a general acid⁄ base mech-anism Previous kinetic and structural studies differ in their interpretation

of the amino acids responsible for the general acid⁄ base mechanism To resolve this discrepancy, we used site-directed mutagenesis and kinetic ana-lysis to study four conserved carboxylic amino acids With the D257A mutant, the Km for Mn2+ and the kcat decreased relative to those of the wild-type by sevenfold and 28-fold, respectively With the E234A mutant, the Km for Mg2+ and l-malate increased relative to those of the wild-type

by 87-fold and 49-fold, respectively, and the kcatremained unaltered, which suggests that the E234 residue plays a critical role in bivalent metal ion binding The kcatfor the D235A and D258A mutants decreased relative to that of the wild-type by 7800-fold and 5200-fold, respectively, for the over-all reaction, by 800-fold and 570-fold, respectively, for the pyruvate reduc-tion partial reacreduc-tion, and by 371-fold and 151-fold, respectively, for the oxaloacetate decarboxylation The activities of the overall reaction and the pyruvate reduction partial reaction of the D258A mutant were rescued by the presence of 50 mm sodium azide In contrast, small free acids did not have a rescue effect on the activities of the E234A, D235A, and D257A mutants These data suggest that D258 may act as a general base to extract the hydrogen of the C2 hydroxy group of l-malate with the aid of D235-chelated Mn2+to polarize the hydroxyl group

enzyme is a general acid that donates a proton in

enol–keto tautomerization [10] However, the crystal

structure of human mitochondrial NAD(P)+

-depend-ent malic enzyme revealed that the oxygen of the

carb-oxy group of D279 (D258 in the pigeon, D295 in

A suum) is structurally too distant to extract the

pro-ton from the C2 hydroxy group of l-malate and would

not play a role in the general acid⁄ base mechanism

[11] Therefore, the authors proposed that K183 (K162

in the pigeon, K199 in A suum) and Y112 (Y91 in the

pigeon, Y126 in A suum) are the general base and

acid, respectively Similar geometry was observed in

A suum mitochondrial NAD-dependent malic enzyme

[12] Recently, Cook and his colleagues [13]

re-exam-ined the contribution of residues Y126, K199, and

D294 (D257 in the pigeon, D274 in the human) to

pH–rate profiles They proposed that these three

resi-dues form a catalytic triad, with K199 as the general

base and Y126 as the general acid in the enzymatic

mechanism

The crystal structure of pigeon malic enzyme showed

that the metal ion is co-ordinated with the carboxylic

group on the side chain of E234, D235, D258, the C1

carboxy group and the C2 hydroxy group of l-malate,

and a free water molecule to form an octahedral

con-formation [14] The metal-binding roles of E234 and

D235 have been confirmed in metal-protected

urea-denaturation studies [15] However, the Km value for

Mn2+ decreased 100-fold with E234Q, but was

unal-tered with D235N This prompted us to examine the

contribution of these three amino-acid residues to

metal ion binding and enzymatic catalysis

In this study, we sought to delineate the possible

roles of these conserved residues in the active site of

pigeon cytosolic NADP+-dependent malic enzyme by

site-directed mutagenesis and detailed enzyme kinetic

studies

Results

Purification and structural characterization of

wild-type and mutant malic enzymes

To evaluate the possible roles of the conserved

carb-oxylic amino acids at the active site, E234, D235,

D257, and D258 in pigeon NADP+-dependent malic

enzyme were replaced by alanine using site-directed

mutagenesis The mutated enzymes were expressed in

Escherichia coli BL21(DE3) and purified All

recom-binant enzymes were shown to be homogeneous by

SDS⁄ PAGE (see Supplementary material Fig S1) CD

spectra of all recombinant enzymes were measured

to evaluate whether the secondary structures of the

mutant enzymes were altered The CD spectra of the four mutant enzymes were very similar to that of the wild-type (Fig S2) Differences in absorption intensity were caused by differences in protein concentration All enzymes had similar contents of a-helix and b-sheet secondary structures Most of the kinetic vari-ation in the mutant enzymes was caused by a lack of functional groups and not by global conformational changes

To determine whether malic enzyme endogenous

to E coli was present in our purified recombinant enzymes, an alternative construct of these mutants was expressed in the pET15b plasmid These mutant enzymes, which contained a His6 tag at the N-termi-nus, were purified using a Ni2+-chelating column to exclude endogenous malic enzyme The enzymatic activities of these constructs were similar to those of enzymes that were purified using an ADP–Sepharose column (data not shown) This suggests that the amount of endogenous enzyme in our preparations was negligible

Steady-state kinetic properties of wild-type and mutant malic enzymes

Preliminary kinetic studies showed that none of the mutants had an appreciable effect on the apparent Km for NADP+ Because the metal ion Km and kcat dif-fered between mutants, we performed detailed initial velocity studies in which both the metal ion and the

l-malate concentrations were varied The kinetic parameters of wild-type and mutant malic enzymes are summarized in Table 1 Replacement of residues D235 and D258 with alanine resulted in Kmvalues for Mn2+ similar to those of the wild-type The kcat values of D235A and D258A were at least four orders of magni-tude less than that of the wild-type enzyme These results suggest that the carboxy groups of D235 and D258 are essential for enzymatic catalysis Of the three metal chelated amino-acid residues (E234, D235, and D258), only the E234A mutant demonstrated a sub-stantial decrease in affinity for bivalent metal ions High concentrations of Mn2+ resulted in the forma-tion of a brownish Mn–malate complex, which inter-fered with the enzyme assay Therefore, Mg2+ was used instead for kinetic studies of the E234A mutant E234A had no effect on kcat, but induced 87-fold and 49-fold increases in Kmvalues for Mg2+and l-malate, respectively The D257A mutant had the least effect

on the Km value for Mn2+ (sevenfold decrease) and the kcat value (28-fold decrease), indicating that the D257 residue is not essential for metal ion binding and catalysis The Km values for the metal ion and the

substrate were similar to the corresponding Kd values

in the wild-type and in all mutants except the D257A

mutant This is in agreement with results showing that

the release of NADPH is the rate-limiting step for

pigeon NADP-dependent malic enzyme [16] The

D257A mutant caused a sevenfold decrease and a

4.5-fold increase in Km and Kd values for the metal

ion, respectively This may be caused by perturbation

of the network of hydrogen bonding in the D257A

mutant [3]

Partial reactions catalysed by recombinant malic

enzymes

The reaction catalysed by malic enzymes consists of

oxidoreduction and decarboxylation The rate of each

reaction can be measured independently of the other

The kinetics of these two reactions were examined in

mutants that decreased kcat of the overall reaction

(D235A, D257A, and D258A) The results of these

studies are summarized in Table 2 The changes in the

kinetic parameters of the D257A mutant were small

relative to those of the D235A and D258A mutants

(fourfold and twofold changes in kcat for

oxidoreduc-tion and decarboxylaoxidoreduc-tion, respectively) However, the

kcat values for both reactions changed substantially

with both the D235A and D258A mutants For the

reduction of pyruvate (the reverse of oxidation of

malate to oxaloacetate), the kcat values decreased

800-fold and 570-fold for D235A and D258A,

respect-ively, and kcatvalues for the decarboxylation of oxalo-acetate decreased 371-fold and 151-fold for D235A and D258A, respectively

pH studies The pH–rate profile of wild-type enzyme showed a bell-shaped curve with pKa values of 6.29 ± 0.01 and 8.78 ± 0.09 at the acidic and basic sites, respectively The pH–rate profiles for D235A, D257A, and D258A also showed bell-shaped curves, with two pKa values (Fig 1) The estimated pKa values from the pH profile studies are summarized in Table 3 The acidic and basic pKavalues for D258A were almost identical with those of the wild-type, and the differences were within the limits of experimental error The acidic pKa values for D235A and D257A were also similar to that of the wild-type, but their basic pKavalues were increased to 9.10 and 9.23, respectively

Chemical rescue experiments Amino-acid residues involved in general acid⁄ base mechanisms can be identified using the chemical rescue method The abilities of the sodium salts of formate, acetate, propionate, butanoate, and azide to rescue lost function of the E234A, D235A, D257A, and D258A mutants were studied None of the small acids rescued the activities of mutants E234A, D235A, or D257A The only restoration of activity occurred with the

Table 1 Kinetic parameters for wild-type and mutant pigeon cytosolic NADP + -dependent malic enzymes.

K mNADP (app)

(l M )

K mMal

(m M )

K dMal

(m M )

K mMn

(l M )

K dMn

(l M )

K mMg

(m M )

K dMg

(m M )

k cat

(s)1) Wild-type

(Mn 2+ )

Wild-type

(Mg 2+ )

Table 2 Kinetic parameters of partial reactions for wild-type and mutant malic enzymes.

K mPyr (app) (m M ) k cat (app) (s)1) K mOAA (app) (m M ) k cat (app) (s)1)

D258A mutant in the presence of azide (Fig 2A).

Activation of D258A reached a maximum at 100 mm

sodium azide and then declined at higher

concentra-tions The extent of activation was underestimated

because of the presence of unsaturated l-malate and

Mn2+in the assay mixture To investigate the

re-acti-vation process further, kinetic parameters for mutant

D258A were determined in the presence of sodium

azide (Table 4) Sodium azide had no significant effect

on Km values for l-malate and Mn2+ and on the kcat value when wild-type enzyme was used With D258A, sodium azide increased the Kmvalues for l-malate and

Mn2+ by 25-fold and 286-fold, respectively, compared with those observed in the absence of sodium azide The kcat value for the D258A mutant was 890 times greater in the presence of the azide ion than in the absence of the azide ion (Tables 1 and 4) The activity

of the D258A mutant was restored to 42% of that of the wild-type by sodium azide To provide further insight into the catalytic roles of the D258 residue, the two partial reactions were examined by azide rescue Only the pyruvate reduction reaction was rescued by sodium azide (Fig 2B) The kinetic studies showed that the kcat value for D258A was identical with that

Table 3 Summary of kcat pH data for wild-type and mutant malic

enzymes.

k cat (s)1)

Fig 1 pH–k cat profiles for wild-type and mutant pigeon cytosolic

NADP + -dependent malic enzyme The profiles for wild-type (s),

D235A (n), D257A (m), and D258A (d) are shown Malic enzyme

activity was assayed as described in Experimental procedures.

Points are the experimental data, and traces are the results of a

fit of data for the pH–rate equation log y ¼ log[C ⁄ (1 + H ⁄ K a1 +

K a2 ⁄ H)].

Fig 2 Fold of activation of mutant malic enzyme as a function of the concentration of sodium azide (A) The mutant malic enzyme overall oxidative decarboxylation activities of E234A (n), D235A (d), D257A (h), and D258A (s) were assayed as described in Experimental procedures (B) The azide rescue of reduction partial reaction of wild-type (s) and D258A (d) and decarboxylation activ-ity of D258A (.).

of the wild-type in the presence of azide (Table 4) The

results of the chemical rescue studies suggest that

D258 may act as a general base to extract the proton

of the C2 hydroxy of l-malate to facilitate

oxaloace-tate formation

Discussion

In these studies, site-directed mutagenesis was used to

evaluate the catalytic roles of four highly conserved

acidic residues in the active site of pigeon NADP+

-dependent malic enzyme Steady-state kinetic

charac-terization of the E234A, D235A, D257A, and D258A

mutants suggests that the D257 residue is not directly

involved in enzyme function E234 is important for the

binding of bivalent metal to the enzyme, and D235

and D258 play critical roles in catalysis

Our kinetic results for the pigeon D257A mutant

differ from those for the corresponding mutant from

A suum The Km and kcat values and the bell-shaped

pH profile of the pigeon D257A mutant did not differ

significantly from that of the wild-type enzyme In

con-trast, the corresponding mutant from A suum, mutant

D294A, had a kcat of about 13 000-fold less than that

of the wild-type and exhibited a pH-independent

pat-tern at the basic end of its pH range [13] The A suum

mitochondrial enzyme is allosterically activated and

inhibited by fumarate and ATP, respectively [17,18],

whereas the pigeon cytosolic enzyme is not regulated

by any known allosteric effector The amino-acid

sequences of these two isozymes show 55% identity

and 73% similarity Therefore, kinetic differences

between pigeon and A suum mutant enzymes are

probably caused by differences in the

microenviron-ments at their active sites

The 3D structure of pigeon malic enzyme showed

that the metal ion was co-ordinated with the carboxy

groups of the E234, D235, and D258 side chains, the

carbonyl group of oxalate (an analogue of

enolpyru-vate), and water to form an octahedral complex [14]

However, our kinetic studies show that only the

E234A mutant has a significant effect on metal

bind-ing These results are consistent with previous studies

in which the metal-binding ability of E234Q was

decreased 100-fold, whereas D235N had little effect on the Km for Mn2+ [20] The unique kinetic properties

of the E234A mutant probably result from the specific geometrical arrangement of E234 The carboxy groups

of E234 and D235 and the C1 carboxy and C2 hydroxy groups of l-malate are coplanarly chelated with Mn2+ D258 and water are located axially above and beneath this plane, respectively In this plane, E234 and D235 are diagonally opposed to the C1 carboxy group of l-malate and the C2 hydroxy group

of l-malate, respectively (Fig 3) The interaction of

Mn2+ and the C1 carboxy group of l-malate should

be strengthened by omitting the chelating of the carb-oxy group of the residue E234 at the opposite direction

in E234A mutant This trans effect will drive the

Mn2+ toward l-malate and therefore decrease the affinity of Mn2+for the carboxy groups of D235 and D258 This may account for the increase in Km when the E234 residue was mutated to alanine The nominal change in Km values observed with the D235A and D258A mutant enzymes may reflect the elimination of

an unfavourable repulsive interaction between the carboxy group and neighbouring negatively charged ligands In previous Fe2+-ascorbate cleavage and site-directed mutagenesis studies, we proposed that D258 was involved in metal ion binding [19,20] However, in those studies, of the four D258 mutants, only D258E

Table 4 Kinetic parameters of overall and reduction partial reaction for wild-type and D258A mutant malic enzyme in the presence of

50 m M sodium azide.

Oxidoreduction decarboxylation of malate reaction Reduction of pyruvate reaction

KmMal(app) (m M ) KmMn(app) (l M ) kcat(app) (s)1) KmPyr(app) (m M ) kcat(app) (s)1)

Fig 3 Proposed mechanism for reduction step of pigeon cytosolic NADP+-dependent malic enzyme The scheme is not meant to imply correct geometry or stereochemistry but simply to show the movement of protons and electrons.

showed any measurable activity Its Km value for

Mn2+ increased by  1600-fold In the present study,

larger amounts of enzyme were used and had a

pro-nounced effect on the kcatvalue but no effect on metal

ion affinity The previously reported effects of D258E

may have been caused by the extra methylene group,

which would have perturbed the position of Mn2+

rel-ative to the other amino-acid residues responsible for

its binding

A significant decrease in the kcatvalue of the D235A

mutant has not been reported previously The carboxy

group of D235, Mn2+, and the C2 hydroxy group of

l-malate are linear with Mn2+ at the centre

There-fore, it is impossible for the D235 residue to act as a

general acid⁄ base in the catalytic mechanism Our

chemical rescue and pH–rate profile results also

sup-port this contention, which is based on crystal

struc-ture It has been proposed that the metal ion acts as a

Lewis acid to stabilize the negatively charged transition

state [21] In D235A, because the interaction between

the carboxy and Mn2+ does not occur, the chelating

ability of Mn2+for the C2 hydroxy group of l-malate

is increased This strong electron-withdrawing ability

might propagate through the C2 hydroxy at the

a-position to the C–H bond at the b-position and

make the hydrogen atom partially positive This effect

might make hydride transfer impossible and inactivate

the enzyme In the wild-type enzyme, this

metal-induced polarization will not extend to the b-position

and will be limited to the C2 hydroxy group of

l-ma-late It will increase the acidity of the hydroxy group

and facilitate the transfer of the proton from the

hydroxy group to the general base residue and the

hydride transfer to NADP+ to complete the

oxidore-duction reaction (Fig 3) The metal ion will then

inter-act with the carbonyl oxygen of oxaloacetate and

facilitate the decarboxylation reaction to form

enol-pyruvate [21] Our kinetic data on the D235A mutant

demonstrated a dramatic decrease in kcatvalues for the

overall reaction and both partial reactions, which is in

agreement with a model in which the metal ion

partici-pates in both partial reactions These results also

indi-cate that both the Lewis acid metal ion and the

general acid⁄ base residue are important for the

cata-lytic mechanism of malic enzyme

The D295 (D258 in the pigeon) residue in A suum

malic enzyme was identified as a general base by

kin-etic and site-directed mutagenesis studies [9] However,

its role has been questioned because of the

inaccessibil-ity of the carboxyl oxygen to the hydrogen of the

hyd-roxy group of l-malate in human [3] and A suum

malic enzymes [11] A similar topology was observed

in pigeon NADP+-dependent malic enzymes, in which

the distance between the carboxy oxygen of D258 and the C2 hydroxy is 3.47 A˚ However, our kinetic studies showed that substitution of alanine for aspartate at the D258 residue decreased kcatvalues in the overall oxida-tive decarboxylation reaction and in the pyruvate reduction partial reaction Both these enzymatic activ-ities of D258A mutant could be rescued by sodium azide No azide rescue was observed for the decarb-oxylation partial reaction These results indicate that the carboxylic group of D258 is essential for the first step of the enzymatic reaction in which a general base

is involved Sodium azide rescue has been widely used

to distinguish nucleophile residues from general bases

in glycosidases, in which azide can act as nucleophile but not as a proton acceptor [22] However, the azide ion was shown to act as an exogenous proton acceptor

in the re-activation of the acid⁄ base mutants of Ther-mobacillus xylanilyticus a-l-arabinofuranosidase [23] and human b-glucuronidase [24] Therefore, despite the contradiction between crystal structure and kinetic studies, we suggest that D258 might still act as a gen-eral base to accept a proton from the C2 hydroxy group to form a ketone and facilitate C2 hydride transfer (Fig 3)

The pH dependence of kcat has been interpreted as ionization of an enzymatic carboxy group essential for catalysis The unexpected bell-shaped pH profile of the D258A mutant indicated that the acidic pKa may derive from chemical components other than the carb-oxy group of the D258 residue The conditions used in the current studies were not acidic enough to reveal the pKa of the carboxy group of l-malate Recently, studies showed that the pKa of the deprotonation of the metal-co-ordinated hydroxy group of isocitrate in the porcine mitochondrial NADP+-dependent isoci-trate dehydrogenase could be shifted to pH 5 [25] Therefore, deprotonation of the metal-chelated hyd-roxy group substrate l-malate may be another reason for the acidic pKain the pH profile

There are several possible reasons for the discrep-ancy between the results of studies of kinetics and those of studies of crystal structure Firstly, the D235 and D258 mutants had the most profound effect on

kcat values This suggests that polarization of the C2 hydroxy group and the general acid⁄ base reaction co-operatively extract the hydrogen of the C2 hydroxy group to facilitate hydride transfer Therefore, the carboxylic group of D258, a weak base because it is relatively distal to the hydroxy group of l-malate, may still be able to act as a general base for the oxidore-duction reaction Secondly, an active-site water mole-cule may exist between the carboxy group of D258 and the hydroxy group of l-malate and serve as a

proton relay to fulfil the general base role of D258.

Similar active-site water molecules have been observed

in the crystal structure of porcine mitochondrial

isoci-trate dehydrogenase, another oxidoreductive

decarbox-ylated enzyme [26] In this case, an aspartate residue

and two water molecules form a catalytic triad that is

responsible for the general base mechanism Finally,

the crystal structures of malic enzyme were solved in

either the E–NADH–malate–Mn2+–fumarate

penten-ary complex (human) or the E–NAD(P)H–oxalate–

Mn2+ tertiary complex (pigeon and Ascarid) Pigeon

and Ascarid malic enzymes show substrate inhibition

in the presence of a high concentration of l-malate

[21,27,28] It has been suggested that the substrate

inhibition might result from the formation of an

E–malate–NADPH–Mn2+ aborted complex Early

kinetic studies showed that oxalate, an analogue of

enolpyruvate, is a dead-end inhibitor for malic enzyme

[29] Therefore, all the 3D structures of the malic

enzyme examined might represent inactive aborted

enzymatic forms The inhibition observed in the kinetic

studies may have resulted from inaccessibility between

the carboxy group of D258 and the C2 hydroxy group

of l-malate Therefore, the carboxy group of D258

may still be close enough to act as a general base to

extract the C2 hydroxy proton of l-malate in the

enzy-matically active complex

In conclusion, we have described the functional roles

of these conserved carboxylic acid amino-acid residues

using site-directed mutagenesis and steady-state

kinet-ics We propose the following:

l E234 is essential for Mn2+binding

l The carboxy groups of D235 and D258 act

co-operatively

l The D235 residue is involved in the polarization of

the hydroxy group of l-malate by chelating the

Mn2+ion

l The D258 residue acts as a general base to promote

oxaloacetate formation and hydride transfer

Experimental procedures

Materials

Restriction endonucleases, T4 DNA polymerase, T4 DNA

ligase, and T4 polynucleotide kinase were purchased from

Promega (Madison, WI, USA) Q Sepharose and

2¢,5¢-ADP–Sepharose were obtained from Amersham

(Piscata-way, NJ, USA) The pET21b expression vector was

purchased from Novagen (Madison, WI, USA) NADP+

was purchased from Sigma (St Louis, MO, USA) All other

reagents were of molecular biology grade or the highest

grade available

Cloning of pigeon liver malic enzyme cDNA

The full-length pigeon liver cytosolic malic enzyme cDNA was cloned into the pET21b vector for expression, as previ-ously described [30] The construction was designed in such

a way that no extra nucleotide sequence flanked the 5¢ end

of the ORF of the malic enzyme cDNA Therefore, the amino-acid composition and sequence of the recombinant form were identical with those of the native enzyme The plasmid containing malic enzyme cDNA was named pET21-ME

Site-directed mutagenesis

Site-directed mutagenesis was carried out according to the procedures of Zoller & Smith [31] using the M13 origin in the vector for uracil-containing ssDNA preparation Other DNA techniques were performed according to the protocols

of Sambrook et al [32] The pET21-ME recombinant phagemid was amplified in the ung–and dut–CJ236 E coli strain with helper phage R408 for preparation of the uracil-containing ssDNA template The uracil-uracil-containing template DNA was annealed with phosphorylated mutagenic oligo-nucleotides and then extended in vitro and ligated by T4 DNA polymerase and T4 DNA ligase, respectively The mutated DNA was screened by transforming into the ung+ and dut+ JM109 E coli strain, and the surviving colonies were further identified by dideoxy chain-termination sequencing [33] The entire cDNA was also sequenced to exclude any unexpected mutations resulting from in vitro DNA polymerase extension

Expression and purification of recombinant malic enzymes

Expression plasmids for wild-type malic enzyme and mutants were introduced into the host E coli BL21(DE3) and grown

in Luria–Bertani medium containing 0.1 mgÆmL)1ampicillin

at 37C to an A660 of 0.5–0.6 Expression was induced with 1.0 mm isopropyl b-d-thiogalactopyranoside The culture was then allowed to grow overnight at 25C The cells were harvested by centrifugation for 15 min at 5000 g Cells were resuspended and sonicated in Tris⁄ HCl buffer (25 mm, pH 7.5) containing 2 mm 2-mercaptoethanol The recombinant proteins were purified using a Q-Sepharose column pre-equilibrated with the same buffer Malic enzyme was eluted with Tris⁄ HCl buffer containing 150 mm NaCl The fractions containing malic enzyme were further purified using a 2¢,5¢-ADP–Sepharose column The malic enzyme was then eluted by 230 lm NADP+ A Sephadex G-25 gel filtration column was used to remove NADP+ All purified enzymes were subjected to SDS⁄ PAGE to examine their purity Protein concentrations were determined by the Bradford method using BSA as a standard [34]

CD measurements

CD measurements were made with a Jasco J-810

spectropo-larimeter using a 0.1-cm path-length cell and averaging five

repeated scans between 250 and 200 nm Typically, 30 lg

of the wild-type or mutated NADP+-dependent malic

enzyme in Tris⁄ HCl buffer (25 mm, pH 7.5) containing

2 mm 2-mercaptoethanol was used for each measurement

The spectra were analysed on DICHROWEB (http://

www.cryst.bbk.ac.uk/cdweb/html/home.html) using the

software of CDSSTR [35,36]

Enzyme assay

Malic enzyme activity was assayed as described by Hsu &

Lardy [37] The reaction mixture contained

triethanol-amine⁄ HCl buffer (66.7 mm, pH 7.4), l-malate (5 mm),

NADP+ (0.23 mm), Mn2+ (4 mm), and an appropriate

amount of enzyme in a total volume of 1 mL The

forma-tion of NADPH at 25C was monitored continuously at

340 nm with a Perkin–Elmer Lambda 3B

spectrophoto-meter One unit of enzyme activity was defined as the

initial rate of 1 lmol NADPH formed per minute under

the assay conditions A molar absorption coefficient of

6.22· 103m)1Æcm)1 for NADPH was used in the

cal-culations Specific activity was defined as lmol NADPH

formedÆmin)1Æ(mg protein))1

Kinetic analysis

Apparent Michaelis constants for the substrates were

determined by varying one substrate concentration around

its Kmvalue while maintaining the other components

con-stant Initial velocity studies were performed to determine

the Michaelis and dissociation constants for l-malate

and Mn2+ For initial velocity studies, the concentrations

of both l-malate and Mn2+ were varied while that of

NADP+was maintained at saturation The E234A mutant

required a higher concentration of Mn2+ for initial

velo-city studies than the other mutants Under these

condi-tions, a brownish Mn–malate complex formed, which

would have interfered with the enzyme assay Therefore,

Mn2+was replaced by Mg2+for initial velocity studies of

the E234A mutant Concentrations of the other

compo-nents were held constant Data were analysed using the

following equation, which describes a sequential initial

velocity pattern:

t¼ VmaxAB=ðKiaKbþ KaBþ KbAþ ABÞ

in which t and Vmaxrepresent initial and maximum

veloci-ties, A and B represent reactant concentrations, Kaand Kb

are Michaelis constants for A and B, and Kiais the

dissoci-ation constant for A The linear regression analysis was

carried out with commercial pro fit 6.0 (QuantumSoft,

Uetikon am See, Switzerland)

Partial reaction analysis

The two partial activities of malic enzyme, decarboxylation and reduction, can be evaluated separately The decarboxy-lation activity of malic enzyme was assayed by the method

of Tang & Hsu [38] using oxaloacetate as substrate The rate of decarboxylation of oxaloacetate was measured by monitoring the disappearance of the enolic oxaloacetate absorbance at 260 nm in the presence of Mn2+or Mg2+ Various concentrations of oxaloacetate in 185 mm potas-sium acetate buffer, pH 4.5, were added to 50 mm EDTA and incubated at 25C for 10 min to reach keto–enol equi-librium The oxaloacetate solutions were added to a total volume of 1 mL containing 4 mm MnCl2and 37 mm potas-sium acetate buffer, pH 4.5 to start the reaction The rate

of decarboxylation in the presence of enzyme was corrected

by subtracting the spontaneous oxaloacetate decarboxyla-tion

Oxidation of l-malate to oxaloacetate cannot be evalu-ated directly because of interference by the subsequent decarboxylation The reversed direction, reduction of a-oxo acid to a-hydroxy acid, can be analysed using pyruvate and NADPH as substrates The reduction partial reaction was performed as described by Tang & Hsu [39] using pyruvate

as substrate The rate of reduction of pyruvate to lactate was measured at 25C by monitoring the decrease in absorbance at 340 mm associated with the oxidation of NADPH A typical assay mixture contained 66.7 mm tri-ethanolamine⁄ HCl buffer (pH 7.4), 0.23 mm NADPH,

4 mm MnCl2, 1–50 mm pyruvate (pH 7.4), and an appro-priate amount of malic enzyme

pH studies

The pH dependencies of kcat for wild-type and mutants were determined using initial velocity studies and variable concentrations of l-malate and NADP+ as a function of

pH over the pH range 5.5–10, which was maintained with

60 mm Bis-Tris propane buffer The pH values were recor-ded and showed no significant change before and after the initial velocity was measured The pKavalues were obtained

by fitting the following equation to the data:

log y¼ log½C=ð1 þ H=Ka1þ Ka2=HÞ

where y is the value of the parameter of interest (kcat), C is the pH-independent value of y, H is the hydrogen ion concentration, and Ka1 and Ka2 are the acid dissociation constants for functional groups in the enzyme–substrate complex

Chemical rescue

The stock solutions of exogenous acids were prepared at

pH 7.4 Various free acids, including formic acid, acetic acid, butyric acid, or sodium azide, were added to the

standard reaction mixture to examine their rescue abilities.

To measure the kinetic properties of malic enzyme after

rescue, 50 mm sodium azide was included for all kinetic

studies

Acknowledgements

This research was supported by a grant from the

National Science Council, China

(NSC92-2320-B016-060 to W.Y.C.) We thank Dr Chi-Ching Hwang

(Kaohsiung Medical University, Taiwan) and Dr

Minghuey Shieh (National Taiwan Normal University,

Taiwan) for helpful discussions

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Supplementary material

The following supplementary material is available online:

Fig S1 SDS-PAGE of purified wild-type and mutant malic enzymes

Fig S2 CD spectra of wild-type and mutated pigeon NADP-malic enzyme

This material is available as part of the online article from http://www.blackwell-synergy.com