Multifunctionality of lipoamide dehydrogenase: Activities of chemically trapped monomeric and dimeric enzymes

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 206, No. 1, January, pp. 77-86, 1981

Multifunctionality of Lipoamide Dehydrogenase: Chemically Trapped Monomeric and Dimeric C. S. TSAI, Department

D. M. TEMPLETON,

Activities Enzymes

of

AND A. J. WAND

of Chemistq and Institute of Biochemistry, Ottawa, Ontario KIS 5B6, Canada

Carleton

University,

Received January 15, 1980 Lipoamide dehydrogenase from pig heart exists in monomer-dimer equilibrium. The effect of the state of subunit aggregation on the multifunctionality of lipoamide dehydrogenase was investigated by the use of chemically trapped monomeric and dimeric yields the enzymes. Reductive carboxymethylation with 2-mercaptoethanol-iodoacetate stable monomeric enzyme which has been isolated for structural and kinetic studies. The chemically induced monomerization is accompanied by conformational changes resulting in an increased mobility of flavin-adenine dinucleotide. The chemically trapped monomer shows an enhanced diaphorase activity, a reduced electron transferase activity, and a complete loss in dehydrogenase as well as transhydrogenase activities. The enhanced diaphorase activity is associated with increased catalytic efficiencies and the reversal of an inhibitory NADH effect at high concentrations. Treatment of lipoamide dehydrogenase with dimethyl suberimidate gives amidinated samples containing crosslinked dimer. The crosslinked enzyme exhibits a higher dehydrogenase catalytic efficiency than the noncrosslinked enzyme with different kinetic mechanisms without significantly affecting the kinetic parameters of diaphorase reaction. Although the dimeric structure is intimately associated with the dehydrogenase activity, it does not preclude the diaphorase activity. An altered flavin-adenine dinucleotide environment accompanying monomerization is likely responsible for the enhanced diaphorase activity.

Flavoenzymes are known to utilize a wide range of substrates with varied chemical structures (1,Z). Lipoamide dehydrogenase (NADH: lipoamide oxidoreductase, EC 1.6.4.3) has been shown to catalyze NADHmediated reversible hydrogenation of lipoamide (dehydrogenase reaction), hydrogen transfer to nicotinamide nucleotides (transhydrogenase reaction), electron transfer to inorganic acceptors (electron transferase reaction), and reduction of quinone dyes (diaphorase reaction). In view of recent interest in multifunctional enzymes (3, 4) this work was initiated to explore experimental parameters which affect multifunctional activities of lipoamide dehydrogenase. Lipoamide dehydrogenase from pig heart consists of two presumably identical subunits. The dimeric enzyme exists in two

interconvertable forms; a dissociable and a nondissociable form (5). A reversible dimermonomer transition has been reported to be facilitated by various means (6). The monomer is characterized by high diaphorase activity and low dehydrogenase activity whereas the opposite characteristics distinguish the dimer. Therefore, any satisfactory explanation concerning the structural and mechanistic aspects of the multifunctional behavior of lipoamide dehydrogenase must consider the dimer-monomer equilibrium and the accompanying structural changes. To gain insight into these processes, we have sought to trap the enzyme in monomeric and dimeric forms by chemical modifications. Such approaches yield a pure nonassociating monomer and preparations containing varied fractions of crosslinked dimer. Kinetic and structural studies using 77

0003-9861/81/010077-10$02.00/O Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form reserved.

TSAI, TEMPLETON,

78

these molecular species are reported and their differential activities to catalyze multifunctional reactions are discussed. MATERIALS

AND METHODS

Materials. Pig heart lipoamide dehydrogenase was obtained from Sigma Chemical Company or BoehringMannheim. These preparations were desalted for use by dialysis against appropriate buffers or by gel filtration in Bio-Gel P-2 columns. The enzyme was checked for purity by electrophoresis and spectrophotometry. When required, further purification was performed on a calcium phosphate cellulose column according to Williams et al. (7). Rabbit muscle aldolase was the product of Sigma Chemical Company and its oligomers were prepared according to Davies and Stark (8). Protein markers for SDS-polyacrylamide gel electrophoresis and sucrose gradient centrifugation were purchased from Worthington Biochemical Corporation. Dichloroindophenol (DCIP), FAD, iodoacetic acid, lipoamide, NADH, thionicotinamide adenosine dinucleotide (TNAD+), and trinitrobenzenesulfonic acid (TNBS) were acquired from Sigma Chemical Company. Dimethylsuberimidate (DMS) and dimethyladipimidate were products of Pierce Chemical Company. Dimethylmalonimidate and dimethylsuccinimidate were synthesized according to the published procedure (9). Sodium dithionite was from Fisher Scientific. 2-Mercaptoethanol was obtained from Eastman Organic Chemicals and [1-YJiodoacetic acid was a product of New England Nuclear. Other chemicals are reagent grade from commercial sources. Preparation of reductive carboxymethylated monomer (Erc,,,). Enzyme solutions (5.0 mglml in 0.10 M potassium phosphate buffer, pH 7.0) were made anaerobic by six cycles of evacuation and flushing with Nz. The enzyme in a quartz Thunberg cell or a sealed Erlenmeyer flask (25 ml) with a side arm was reduced by an addition of 20 ~1 2-mercaptoethanoli ml of enzyme solution on ice. After 30 min, an appropriate amount (50 mg/ml) of iodoacetic acid was added from the side arm into a foil-wrapped vessel. The reaction mixture was placed in the dark for 30 min and the reaction was stopped by the removal of reagents on a Bio-Gel P-100 column which simultaneously separated monomeric and dimeric protein species. Controls were prepared in an identical manner by omitting 2-mercaptoethanol and/or iodoacetic acid. ’ Abbreviations used: ATR, attenuated total reflectance; DMS, dimethyl suberimidate; DCIP, dichloroindophenol; EDTA, sodium salt of ethylenediaminetetraacetic acid; E,,, reductive carboxymethylated monomeric enzyme; E,,,, crosslinked (DMS) dimeric enyzme; SDS, sodium dodecyl sulfate; TNBS, trinitrobenzenesulfonic acid; TNAD+, thionicotinamide adenosine dinucleotide.

AND WAND The concentration of monomer was determined by the microbiuret assay for proteins (10). The incorporation of radioactivity from [ l-14C]iodoacetic acid was followed by withdrawing aliquots at time intervals. These aliquots were diluted with water, centrifuged in Centriflo membrane cones (CF 25, Amicon Corp.), washed several times with water, and air dried. The dry cones were cut and the radioactivity was counted in a Beckman LS150 scintillation counter, using standard 14Cquench curve corrections. Preparation of cross-linked dimer (E,,,). To the enzyme solution (2.0 mgiml) in 0.10 M N-ethylmorpholine’HC1 buffer, pH 8.5 (or 0.10 M triethanol amine buffer, pH 8.5), was added an equivalent volume of DMS (l-3 mg/ml) in the same buffer. The reaction mixture was stirred at room temperature for desired time periods to obtain preparation of various degrees of crosslinking. At the end of reaction, the reagent was removed by Sephadex G25 filtration or dialysis against 0.50 mM phosphate buffer, pH 7.0, containing 0.30 mM EDTA. The analyses of E,,, in amidinated samples were carried out. by SDS-polyacrylamide gel electrophoresis. The noncrosslinked protein in the samples was dissociated by incubation (2 h, 37°C) in 8.0 M urea containing 0.10% SDS and 0.10% 2-mercaptoethanol. Gel electrophoresis was carried out as described (11). Protein bands were visualized with Coomassie brilliant blue and the gels were scanned using a Beckman gel scanner (Model 198402) attached to Acta C III spectrophotometer at 600 nm. Peak integration of the densitometer tracings was performed with a Keuffel and Esser compensating polar planimeter. Enzyme assays and kinetic studies. All enzyme assays and kinetic studies were carried out in 50 mM potassium phosphate buffer, pH 7.0, by means of a Perkin-Elmer spectrophotometer (Coleman Model 124) equipped with a variable output recorder (Coleman Model 165) and a thermostat circulator maintained at 25 t 0.5”C. Dehydrogenase, transhydrogenase, electron transferase, and diaphorase activities were assayed by the use of 0.50, 0.50, 5.0, and 5.0 pg of enzyme, respectively, in a final volume of 1.0 ml assay mixture containing 50 pM of NADH plus 250 pM of lipoamide, 125 pM of TNAD+, 250 pM of K,Fe(CN),, or 25 NM of DCIP, respectively, at 340, 395, 420, or 600 nm. Kinetic studies of dehydrogenase and diaphorase of chemically trapped enzymes were carried out as described (12). The concave region was analyzed according to the publighed method (13). Since we could not obtain pure E,,, either by prolonged amidination or improved isolation, kinetic studies of the crosslinked enzyme were performed with enzymatic preparations containing varied fractions (X) of E,,,. The observed initial rates (v) which showed biphasic reciprocal relationship with substrate concentrations were treated as a linear com-

LIPOAMIDE

DEHYDROGENASE TABLE

79

MULTIFUNCTIONALITY I

ACTIVITIES OF REDUCTIVE CARBOXYMETHYLATED MONOMER (EreM) Activities (v/E, x 10e3 min-I) Dehydrogenase Transhydrogenase Electron transferase DPase diaphorase

Native

Control HS(CH&OH

Control ICH&OZH

E r&i

2.8 1.2 1.2

2.7 + 0.3 1.3 2 0.1

0.35 t 0.2 0.3 + 0.2

0 0 0.25

0.040

0.04 * 0.005

0.025

k 0.01

0.25-1.3

Note. Data for the native enzyme and E,, (FAD:EwM = 0.40 ? 0.15) are averages of more than ten experiments while those for controls are averages of two experiments. Activities were expressed as v/E, where v and E, are initial rate and enzyme concentration, respectively.

bination of rates contributed by X fraction of the crosslinked enzyme (E,) and (1-X) fraction of the noncrosslinked enzyme (EN): ’ = AB

V,AB + K,,A

+ K,,B +

AB

+ K,,B

f KlaxKbx V,AB + Kb,A

+ K,,,K,,

’

111

where subscripts x and N denote kinetic parameters for the crosslinked and noncrosslinked enzymes. V, K,, Kb, and Ki, are maximum velocity, Michaelis constant for NADH, lipoamide, and inhibition constant for NADH, respectively. By introducing k, = VJ E, with E, = XE, and kN = VdEN with EN = (1-X) E,, Eq. [l] can be transformed into v - = VN + (v, - YN)X, Et

PI

where = AB

+ K,,B

k,AB + K,,A

vN = AB

+ K,,B

k,AB + K,,A

”

+ KiaxKbx

’

+ K,,,K,,

’

131 141

Plots of v/Et versus X at different concentrations of A and B provide Y, and Ye which are analyzed according to Eqs. [3] and [4] double reciprocally by the graphical method analogous to that of Dalziel (14) to give kinetic parameters for the crosslinked and noncrosslinked enzymes. Spectroscopic methods. Ultraviolet-visible spectra were obtained on a Cary-14 spectrophotometer using a changeable slide wire for full scale deflections corresponding to O-1.0, O-0.5, or O-O.1 absorbance units. Infrared spectra were recorded on a PerkinElmer Model 225 Grating infrared spectrophotometer by the method of attenuated total reflectance (ATR) (15). Protein samples (l-2 mg in aqueous solution) were layered on polyethylene squares cut to fit the ATR crystals (KRS-5 crystals or IRTRAN 4 crystals). They were dried in an evacuated dessicator over

P,Os overnight and sandwiched against the ATR crystals. The assembled sample was placed in an RIIC TR-25 ATR minor assembly (Beckman) for recording the spectra. Other analytical methods. Amino acid analyses were performed on 0.5mg samples of protein following removal of flavin according to Veeger and Visser (16). A Beckman automatic amino acid analyzer was used. Reactive e-amino groups were determined colorimetrically by titration with TNBS according to Fields (17). SDS-polyacrylamide gel electrophoresis for the estimation of molecular weight was performed according to Weber et al. (11) with 0.10 M borate buffer, pH 3.5, containing 0.10% SDS. Sucrose gradient centrifugation was carried out after the method of Martin and Ames (18). RESULTS

To correlate the subunit structures with multifunctional activities of lipoamide dehydrogenase, attempts were made to prepare stable monomeric and dimeric enzymes trapped in chemically modified forms. Reductive carboxymethylated lipoamide dehydrogenase (ErcM) prepared by the anaerobic 2-mercaptoethanol-iodoacetic acid treatment sequence shows an increased diaphorase activity, a reduced electron transferase activity, and a complete loss of dehydrogenase and transhydrogenase activities (Table I). The degree of diaphorase enhancement was rather variable. The control with 2-mercaptoethanol shows no detectable effect on multifunctional activities. However a reduction in all measured activities follows extensive carboxymethylation without the thiol pretreatment. Figure 1 shows that an increase in diaphorase activity with concomitant loss

80

TSAI, TEMPLETON.

JuY77-J 20

25

Time(min.1

FIG. 1. Progress curves of [“CJiodoacetic acid incorporation and changes in DHase and DPhase activities. Addition of [1-‘%]iodoacetic acid to 2mercaptoethanol pretreated enyzme under N, defines zero time. (0), Moles “C incorporated per mole monomer; (O), specific activity in diaphorase reaction; (O), specific activity in dehydrogenase reaction. v and E, are initial rates and total enzyme concentration, respectively.

of the dehydrogenase activity is complete by 5 min, at which time, incorporation of [lJ4C]iodoacetic acid is also at a maximum. The use of [ 1-14C]iodoacetic acid reveals uptake of 21-22 mol of iodoacetate per mole of enzyme-bound flavin. Amino acid analysis indicates, however, the modification of a total of 15-16 groups per reductive carboxymethylated monomer consisting of cysteine, tyrosine, and methionine residues (Table II). A higher value for the 14C incorporation may arise from the analytical method adapted for following 14C carboxymethylation in the presence of Z-mercaptoethanol by the use of Centriflo membrane centrifugation. In the iodoacetic acid control, all cysteine and half of methionine residues are modified. With 2-mercaptoethanol pretreatment, however, approximately two cysteine residues per monomer are modified by reductive carboxymethylation, while all methionine residues are preferentially modified. On elution from a Bio-Gel P-100 column (1.0 x 35 cm), the native enzyme gives two A,,, absorbing peaks. The major peak (more than 90% of the total protein) corresponds to a protein with molecular weight above the exclusion limit of the column (>105 g mol-*). An identical elution profile was observed for the iodoacetate-treated control

AND WAND

enzyme. When E,,, sample was passed through the column, all but a trace of the protein appeared at an elution volume corresponding to the second peak, separated cleanly from the heavier dimer but poorly resolved from the labilized flavin chromophore (Fig. 2). Use of a shorter Bio-Gel P-100 column (1.0 x 16 cm) gave rise to similar elution profiles with respect to the separation of monomer and dimer but resulted in the monomer more heavily associated with flavin. Due both to the requirement of FAD for catalytic activity and its stabilizing effect for the monomer as discussed below, E,,, is routinely purified using the shorter column. The gel filtration chromatography has established a molecular weight for E,,, corresponding to that of the native lipoamide dehydrogenase monomer. In addition, sucrose gradient centrifugation reveals native enzyme components of 1.01 x lo5 and 5.9 x lo4 g mol-’ while ErcM has an apparent molecular weight of 4.6 x lo4 g mol-l. SDS-polyacrylamide gel electrophoresis also shows a single band for E,, with a molecular weight corresponding to 4.8 x lo4 g mol-‘. Under no circumstances was any dimerization of ErcM obTABLE

II

AMINO ACID RESIDUES REDUCTIVE

WHICH ARE MODIFIED CARBOXYMETHYLATION

BY

Residues remaining

Amino acids modified Cysteine + H-cystine Methionine Tyrosine

Native 10 10

7

Control ICH,CO,H

E ret4 (reductive carboxymethylation)

1.8 5 6.7

7.5 0 4.5

Note. Values which are averages of three determinations (FAD/E,,, = 0.40 ? 0.15) indicate the number of moles of the affected residues remained. In reductive carboxymethylation 2-mercaptoethanol protects 5-6 sulfhydryl groups from carboxymethylation likely via formation of mixed disulfides. A total of 15 groups consisting of cysteine, methionine, and tyrosine are modified.

LIPOAMIDE

DEHYDROGENASE

served, as detected either by gel filtration, sucrose gradient centrifugation, SDS-gel electrophoresis, or the return of dehydrogenase and transhydrogenase activities. Although flavin is not tightly associated with modified protein, its separation is not clean. Complete removal of FAD from ErcM by dialysis is relatively facile contrary to the situation with native dimer. The extensive separation of flavin from the modified monomer on the longer column or dialysis leads to decreases in electron transferase and diaphorase activities suggesting the requirement of FAD for the activities of E,,. Both diaphorase and electron transferase activities of Ercb, increase to maximum value at a FAD:monomer ratio of 1:l without further increase upon addition of excess FAD (Fig. 3). At relative FAD concentrations of less than 0.3, activity is lost due to visible precipitation of protein during dialysis prior to the complete removal of FAD. In each case, the activities extrapolate to near zero at zero FAD concentration. Reductive carboxymethylated monomer that lacks visible FAD absorption can be obtained by the use of a longer (35 cm) Bio-Gel P-100 column. On incubation with equimolar or excess FAD at room temperature for 30 min, the spectrum of bound FAD is discernible by the appearance of a slight shoulder at ca. 465 nm. Infrared spectra as shown in Fig. 4 furnish evidence 0.41

I

03

* t

f\

* o.2t I I

ELUTION

I

VOLUME

(ml)

FIG. 2. Elution patterns of the native enzyme (A) and E,, (B) from a Bio-Gel P-100 column (1.0 x 35 cm). Protein content (-) and FAD content (- - -) were monitored spectrophotometrically at 280 and 450 nm, respectively.

81

MULTIFUNCTIONALITY

O--’

[FAD]

/ [ Monomer]

5

FIG. 3. Effect of FAD on the activity of E,,,. Diaphorase (0) (x 10-l) and electron transferase (0) activities were assayed in the presence of varied concentrations of added FAD.

supporting the conformational changes in E rcM. The relative intensities of 1630 cm-’ and 1650 cm-’ component peaks of the Amide I (v,=J band of ErcM are reversed as compared to those of the native enzyme, and the 1500-1550 cm-’ Amide II (yNmH) band (19) is broadened. Both these observations are indicative of a higher a-helical content in the modified monomeric enzyme in agreement with the apoenzyme monomer which has a higher a-helical content than the native dimeric enzyme (16). The TNBS assay indicates a 30 t 5% increase in the number of exposed e-amino groups accompanying the conformational changes of E rcM over both native and iodoacetatetreated control enzymes. The reductive carboxymethylated monomer was subjected to kinetic studies. Whereas marked substrate inhibition of the diaphorase reaction in excess of 0.15 -r- 0.05 mM NADH is observed with the native enzyme, the substrate activation by high NADH concentrations characterizes E TCM(Fig. 5). Therefore, detailed kinetic analysis for diaphorase reactions were carried out in both asymptotic and concave regions (13). Table III shows that the enhanced diaphorase activity of ErcM is associated with increased maximum velocities (V and V’), and increased Michaelis

82

TSAI, TEMPLETON,

AND WAND

(b)

I

I

1800

1700

1600

1500

I400

1700 I/ (cm-’

1600

1500

1400

1

FIG. 4. Attentuated total reflectance infrared spectra of (a) native enzyme and (b) E,,, were taken as described in the text.

constants as well as inhibition constants for NADH (K,, KL as well as Ki,, K&) in both regions. The transition from NADH inhibition for the native enzyme to NADH activation for ErcM arises from large increases in V' and K:,. Complementary to the preparation of a stable monomeric enzyme, we have attempted to prepare a covalently crosslinked dimer. Of a number of crosslinking reagents tested, bifunctional imidoesters offered a promise. The formation of crosslinked dimer was verified by SDS-gel electrophoresis under the condition that the

0

1 IO [NAoH2;-l

I

I

I

30

40

50

(mM-‘1

FIG. 5. Reciprocal plots of initial rate for DPase reactions of [DCIP] = 0.010 rnM showing NADH inhibition of the native enzyme (0) (X 10el) versus NADH activation of E,, (0) at high concentrations.

noncrosslinked enzyme would have been completely dissociated. Figure 6 shows the variation in the effectiveness of diimidoesters with various chain length to crosslink lipoamide dehydrogenase. Repeated efforts to increase the yield of the crossTABLE KINETIC

PARAMETERS

III

FOR DIAPHORASE

REACTION

Kinetic region

Kinetic parameters

Native

Asymptotic

V (@A mir-’ ) Ka (PM) Kb (PM) K, (PM) V/E, (min-I)

16.7 10.8 46.7 7.64 335

370 63.5 25.7 19.5 13.5 x 103

Concave

V’ (PM mix-‘) K (PM) Kb (PM) Kh (PM) %a, (CL@ VI/Et (mir-‘)

14.3 34.3 61.1

0.98 x 103 133 37.5 10.7

1.67 715

E,,

48.8 x 101

Note. V, K,, Kb, and K, are maximum velocity, and Michaelis constants for NADH, DCIP, and inhibition constant for NADH, respectively, in the asymptotic region. Prime indicates the corresponding kinetic parameters in the concave region except composite constants, Kg, and KL,, which are defined according to Tsai (13) such that Kh,, is associated with activation whereas KL,, , with inhibition.

LIPOAMIDE

DEHYDROGENASE

83

MULTIFUNCTIONALITY

FIG. 6. Chain length effect on crosslinking of lipoamide dehydrogenase. Lipoamide dehydrogenase was treated with diimidoesters,

+NH, II CH,-0-C-(CH,),-C-OCH,

NH, II

for 3 h. The amidinated samples were subjected to SDS-gel electrophoresis which separated crosslinked dimer and noncrosslinked enzyme under dissociation conditions. The molecular size-mobility relationship was calibrated with aldolase oligomers (8) in a parallel run. The percentage crosslinkage was estimated from areas under peaks of densitometer scans. The diimidoesters used and corresponding linkages are: (a) control, 0%; (b) dimethyl imidosuccinimidate (n = Z), 16%; (c) dimethyl imidoadipimidate (n = 4), 2’7%, and (d) dimethyl imidosuberimidate (n = 6), 59%. Dimethyl imidomalonimidate (n = l), 17%, was not shown. Abscissa shows distance from the dye front (anode) in arbitrary units.

linked dimer (E,,,) or to isolate the pure EclD were unsuccessful. Therefore the time course for crosslinking with DMS was constructed as the guide for preparing amidinated samples with varied fractions of Ecm to be used in the subsequent studies. Amino acid analysis indicates that amidination with DMS specifically modifies lysine residues. No discernible spectral changes were observed for the amidinated samples. Enzymic assays show a slight general decline in multifunctional activities of the amidinated samples, which contain both EelD and the noncrosslinked amidinated enzyme (Table IV). Kinetic studies were carried out to investigate dehydrogenase and diaphorase activities of EclD by the linear combination of initial rates of amidinated samples with 0, 18 + 1, 32 21, and 42 t 1% EclD. Initial rates were plotted against fraction (X) of EclD to obtain v, and vuNwhich were plotted reciprocally versus NADH or lipoamide in dehydrogenase (Fig. ‘7) or DCIP in diaphorase reactions, respec-

tively. This approach yields kinetic parameters for the crosslinked and the noncrosslinked amidinated enzymes, respectively, from the kinetic studies of its mixtures. A visual examination of the plots for the dehydrogenase reactions shows the contrast between the converging reciprocal plots of sequential mechanism for the crosslinked TABLE ACTIVITIES

OF AMIDINATED Percentage

Percentage Em in amidinated samples 15 30 45

IV

Dehydrogenase 94.6 88.2 85.5

activity

TrSJl.9 hydrogenase 98.9 97.8 94.6

SAMPLES of the control Electron transferase

Diaphorase

94.6 92.1 87.3

96.3 92.6 88.9

Note. Lipoamide dehydrogenase was treated with DMS for different time periods to produce amidinated samples with varied fractions of E,,, which was estimated by SDS-gel electrophoresis. Total assay activities (including crosslinked and noncrosslinked enzymes) are given.

84

TSAI,TEMPLETON,ANDWAND

2.0

m

1.5

b x 1.0 --

.5 E

'9

.5

2.5

y 1.5Q x

25

30

D

35

300 [ NADH

I-’

(mu-’

350

)

FIG. 7. Kinetic analyses of DHase reaction by linear combination of initial rates. (A) Specific initial rates (v/E,) as a function of the fraction of crosslinkage (X) at [NADH] = 5.34 pM and [lipoamide] = 62.4 pM. These data were used to obtain values of vx and I+ according to Eq. [2]. (B) Double reciprocal plots of v;’ vs [lipoamidel-’ according to Eq. [3]. Values of V, were obtained with Eq. [2] at [NADH] = 2.92, 5.34, 10.2, 14.6, and 29.3 FM. Visual analyses of the data points show converging lines of E,.,,. (C) Double reciprocal plots of vi’ vs [lipoamidel-’ according to Eq. [4]. Values of Ye were obtained with Eq. [2] at [NADH] = 2.92, 5.84, 10.2, and 14.6 pM. Visual analyses of the data points show parallel lines for noncrosslinked enyzme. (D) Secondary plots of intercepts (0, A) and slopes (0, A) from (B) and (C) vs [NADH]-‘. Kinetic parameters for E,,, (0, 0) and noncrosslinked enyzme (A, A) were evaluated from these plots and summarized in Table V.

E,,, and parallel reciprocal plots of Ping Pong mechanism for the noncrosslinked amidinated enzyme (Figs. ‘7B vs C) as well as the native enzyme (12). Kinetic parameters for EclD and the noncrosslinked enzyme are summarized in Table V. The crosslinking enhances the catalytic efficiency (VIE) of dehydrogenase reaction without affecting kinetic parameters of diaphorase reaction. A slight decline in dehydrogenase activity of the amidinated samples assayed during amidination must be due to the noncrosslinked enzyme, which has lower catalytic efficiency than E,,, and the native enzyme.

DISCUSSION

Lipoamide dehydrogenase is a tlavoenzyme which exists in a monomer-dimer equilibrium. Among the multifunctional reactions catalyzed by pig heart lipoamide dehydrogenase, dehydrogenase activity was stipulated to associate with the dimer while diaphorase activity was found to be favored by the monomer (20). However the molecular mechanism of these effects has not been elucidated due to the difficulties in obtaining pure stable monomeric and dimeric enzymes. Attempts were therefore made to prepare chemically trapped

LIPOAMIDE

DEHYDROGENASE

stable monomeric and dimeric enyzmes for investigating their structural and mechanistic implications. We have prepared the stable reductive carboxymethylated monomer (ErcM) and the amidinated samples containing crosslinked dimer (E,,,). Dissociation to the monomer by reductive carboxymethylation greatly labilizes FAD; however, the flavin seems to be loosely associated with E,,, since the monomeric enzyme is still reduced anaerobically by NADH resulting in a rapid bleaching of flavin characteristic of bound FAD. Furthermore reconstitution experiments show that 1 mol of flavin per mole of ErcM is necessary and sufficient for maximum catalytic activity. This indicates a specific, single-site interaction rather than a rapid turnover of flavin, as has been suggested for the residual activity of the monomeric apoenzyme of the unmodified enzyme (‘7). Extensive conformational changes resulting in a higher a-helical content and labilization of bound FAD are seen to accompany monomerization. This may result in a stable monomeric structure. On the other hand, the dissociation of FAD causes precipitation of the monomer, therefore the association of FAD is required for the formation of the stable and soluble E,,,. The reductive carboxymethylated monomer completely loses the dehydrogenase and transhydrogenase activities but retains the residual electron transferase activity with greatly enhanced diaphorase activity. For the diaphorase activity, the native enzyme is subject to NADH inhibition whereas ErcM is characterized by NADH

v=

TABLE

V

KINETIC PARAMETERS FOR DEHYDROGENASE AND DIAPHORASE REACTIONS CATALYZED BY CROSSLINKED DIMERIC (E,,,) AND NONCROSSLINKED AMIDINATED ENZYMES

Kinetic parameters

Crosslinked dimer, E,,,

Noncrosslinked amidinated enzyme

DHase V/E (mix]) K, (PM) Kb (PM) Ki, (PM)

18.4 x 103 11.0 217 15.1

7.27 x 103 22.9 358

DPase V/E (min-I) K, (PM) K, (PM) &a (ILM)

980 23.5 40.5 23.1

996 20.4 71.7 14.9

Note. Linear combination of initial rates simultaneously solves for kinetic parameters of twocomponent enzymic species in amidinated samples with known fractions of E,,,. Detailed analyses for DHase reaction are elucidated in Fig. 7. Identical analyses yield kinetic parameters for DPase reaction. E represents E, for E,,, or EN for the noncrosslinked amidinated enzyme, respectively.

activation. These observations can be explained by a common mechanism, the random sequence with a Ping Pong loop which has been proposed for lipoamide dehydrogenase-catalyzed reactions (12). Diaphorase reaction favors the flux of reactants through the random mechanism which is described by the concave converging initial rate behaviors (13, 21) according to the rate equation:

(n,,AB + na,bA2B + nabzAB2)Et d, + d,A + dbB + d,,,AB + d,,A2 + c&B2 + d,,bA’B

where nsi and d,, are kinetic coefficients for numerator and denominator terms, respectively, associated with substrate x (A or B) of the ith degree (13). The NADH inhibition of the native enzyme results from (nab + nab&.(& + da,& > nadda + dd + dabnB2) whereas the NADH activation of E TCM implicates n&da + dabB + d,,,B”) * (nab + nabnB)(dap + d,,,,B). This gives rise to the reversal in K&. The enhanced

85

MULTIFUNCTIONALITY

+ dab,AB’

’

diaphorase activity of ErcM is associated with a large increase in catalytic efficiencies (VIE, and V’IE,) though increases in K, and Kiay which partially neutralize the VIE, effect. It implies that the monomerization reduces the affinity of the enzyme for nicotinamide coenzyme and facilitates the oxidation of reduced FAD by DCIP. The application of the linear combination

86

TSAI, TEMPLETON,

of initial rates enables us to evaluate kinetic parameters for both the crosslinked dimer, E,,,,, and the noncrosslinked amidinated enzyme from the kinetic studies of amidinated samples with varied fractions of crosslinkage. Since we were unable to obtain pure EClo, kinetic mechanisms for EclD and the noncrosslinked enzyme were inferred from initial rate studies and the mechanism proposed for the native enzyme (12). The crosslinking does not seem to affect kinetic parameters of diaphorase reactions. However, EclD catalyzes dehydrogenase reaction via a sequential mechanism notwithstanding the preference of a Ping Pong mechanism (12) for the native and noncrosslinked enzymes. Lipoamide dehydrogenase catalyzes multifunctional reactions via the mixed random Ping Pong mechanism (12), which is simplified to either Ping Pong or ordered BiBi mechanism in the asymptotic region depending on the fluxes of reactants through the competing pathways. A slow conversion of the free enzyme E to the reduced enzyme intermediate, EH2, in comparison to the rate of ternary complex (EAB) formation may contribute to a shift in kinetic mechanism for EClo. Michaelis constants for the crosslinked and noncrosslinked enzymes are not significantly different. However, the catalytic efficiency (VIE) of EclD is higher than the noncrosslinked amidinated and the native enzymes which exist in monomerdimer equilibrium. This is in agreement with the previous report that the dehydrogenase activity is associated with the dimeric species (19). Since the formation of the crosslinked dimer does not affect kinetic parameters of diaphorase reaction, the increased catalytic efficiency of the diaphorase reaction for the monomeric enzyme may arise from the labilization of FAD and accompanied conformational changes. The crosslinking dimerization facilitates the ternary complex formation favoring the ordered BiBi mechanism for the dehydrogenase reaction with a higher catalytic efficiency. ACKNOWLEDGMENTS This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. We thank Dr. J. M. Neelin, Department

AND WAND of Biology, Carleton University, for providing gel scanning facility and Dr. M. Yaguchi of the National Research Council of Canada for amino acid analyses. D.M.T. is a holder of NSERC graduate scholarship and A.J.W. is a holder of Ontario Graduate scholarship. REFERENCES 1.

DIXON, M. (1971) Biochim. Biophys. Acta

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