Multifunctionality of lipoamide dehydrogenase: Promotion of electron transferase reaction

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 225, No. 2, September, pp. 554-561, 1983

Multifunctionality of Lipoamide DehydrogenaselS2: Promotion of Electron Transferase Reaction C. S. TSAI, Lkpartnwnt

of

A. J. WAND,

Chemistry and Institute Received

October

D. M. TEMPLETON, of

Biochemistry,

Carl&m

14, 1982, and in revised

form

AND

P. M. WEISS

University, Ottawa, Canada April

KlS 5B6

18, 1983

Various approaches to promote the one-electron transfer reaction of lipoamide dehydrogenase have been investigated. An addition of riboflavin facilitates the electron transfer between NADH and Fe(CN)i-. Aminocarboxymethylation and cadmium derivatization of the catalytic disulfide moderately activate the electron transfer reaction. An enhancement in the electron transferase activity of the Co(II)-enzyme complex is associated with decreased Michaelis and inhibition constants. Phosphopyridoxamidation identifies the suppressive effect on the electron transferase activity of carboxyl groups proximal to the catalytic histidine residue of lipoamide dehydrogenase. Amidation of these carboxyl groups with diamine greatly promote the one-electron transfer reaction. The increased electron transferase activity of the amidated enzyme is related to the charge nature of the amidated nucleophile and associated with the increased catalytic efficiency which undergoes a shift in the pH profile. The introduction of cationic aminoethyl groups presumably encourages the formation of an anionic flavosemiquinone which promotes the one-electron transfer reaction.

Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) from pig heart is a flavoenzyme which catalyzes NADH-linked reversible reduction of lipoamide (RDase), reversible transhydrogenation between nicotinamide nucleotides (THase), electron transfer to inorganic acceptor (ETase) and reduction of quinone dyes (DPase). These multifunctional activities (1) are a result of the ability of reduced flavin to promote one-electron and two-electron transfers to different acceptors of varied structures (2). The electron

transferase reaction which transfers the reduced equivalent to ferricyanide via oneelectron mechanism is particularly interesting, since the physiological reductase reaction of lipoamide dehydrogenase proceeds presumably via two-electron pathway (3,4). A knowledge of factors affecting the electron transferase reaction would be undoubtedly valuable to our understanding of the multifunctionality and redox chemistry of lipoamide dehydrogenase. Different approaches to enhance the electron transferase activity are described and their implications are discussed.

i Taken in part from the Ph.D. thesis of DMT and M.Sc. thesis of AJW submitted to the Faculty of Graduate Studies and Research, Carleton University. a Multifunctional activities of l&amide dehydrogenase refer to reductase (RDase) assayed by NADH + D,L-lipoamide, transhydrogenase (THase) assayed by NADH + TNAD+, electron transferase (ETase) assayed by NADH + Fe(CN#-, and diaphorase assayed by NADH + 2,6-dichloroindophenol. 6903-9861&l

83.00

Copyright0 1983by AcademicPress.Inc. All rights of reproductionin anyform reserved.

EXPERIMENTAL

PROCEDURES

Materials. Pig heart lipoamide dehydrogenase was obtained from Sigma Chemical Company or Boehringer-Mannheim. Prior to use, the enzyme was checked for purity (5) and desalted by dialysis against appropriate buffers or by Bio-Gel P6 filtration. Enzyme concentrations were determined at 455 nm using an extinction coefficient of 11.3 rn& cm-’ and cal554

ONE-ELECTRON

TRANSFER

REACTION

culated on the basis of FAD content. D,L-Lipoamide, NAD+, NADH, thionicotinamide adenine dinucleotide (TNAD+)3, pyridoxine&phosphate, pyridoxal-5’phosphate (PLP): pyridoxamine-5’-phosphate (PNP) were provided by Sigma Chemical Company. Riboflavin, propylamine, ethylene diamine, and 2-aminoethane sulfonic acid were purchased from EastmanKodak Company. Potassium ferricyanide, 2,6-dichloroindophenol, CdClx, and CoSO, were supplied by Fisher Scientific Company. 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was the product of Pierce Chemical Company. Absolute ethanol was supplied by Consolidated Chemical Company. Bio-Gel P6 was purchased from Bio-Rad Laboratories. Cobalt chelation. The procedure of Huang and Brady (6) was modified to accommodate the instability of lipoamide dehydrogenase for a prolonged exposure to a high pH at room temperature. Lipoamide dehydrogenase was dialyzed overnight against 50 mM borate buffer, pH 10, at 4°C. Upon warming to room temperature, the enzyme solution (l-2 mg ml-‘, pH 10) in a Thunberg tube was immediately made anaerobic by six cycles of deaeration followed by flushing with Nz gas. The anaerobic enzyme was reduced to EHp by addition of a twofold excess of NADH. A twofold excess of CoSO, was immediately added. After 5 min in room temperature, the excess reagents were removed by dialysis against 0.10 M phosphate buffer, pH 7.0, at 4°C which also transfer the Co(H)-enzyme complex to a neutral PH. Amid&on The amide formation between carboxyl residues of lipoamide dehydrogenase and amine nucleophiles followed the published procedure (7). The enzyme was desalted by filtration through a Bio-Gel P6 column and dialyzed against deionized water (adjusted to pH 5.5) exhaustively. Amidation was initiated by the addition of ‘/ volume each of 0.20 M EDC and 0.20 M amine nucleophile in deionized water (pH 5.5) to the enzyme solution (1.0 mg ml-‘) at pH 5.5 by means of a pH stat at 25 + 5°C to give the final enzyme concentration of 0.50 mg ml-‘. The amidation was followed by monitering RDase and ETase

a Abbreviations for modified lipoamide dehydrogenase used: EHz, half-reduced form; EH,, fully reduced form; E,,, aminocarboxymethylated enzyme; Ec.,, Co(I1) complex; Ear cadmium derivative; E,,, pyridoxine+‘-phosphate-treated enzyme; EpLp, phosphopyridoxalated enzyme; Erupt phosphopyridoxamidated enzyme; EN&. amide of HaN (CH,), NH;; EC&, amide of H,N (CH,), CH,; Es,, amide of HzN (CHz)zSO-“. Abbreviations for chemicals included: EDC, l-ethyl-3(3-dimethylaminopropyl) carbodiimide; PLP, pyridoxal-5’-phosphate; PNP, pyridoxamine-6phosphate; TNAD+, thionicotinamide adenine dinucleotide.

OF

LIPOAMIDE

DEHYDROGENASE

555

activities. The reaction was quenched with an equal volume of 0.10 M phosphate buffer, pH 7.5, and dialyzed immediately. Phosphopyridoxamidation was carried out in an identical manner by the use of PNP as the nucleophile. Controls were prepared in parallel experiments by omitting amine nucleophiles which affected neither RDase or ETase activities during amidation. EDC stimulated ETase activity slightly. Other chemical modifications Phosphopyridoxalation was performed as described (8) by reacting lipoamide dehydrogenase with PLP. The reaction was quenched with NaBHd. An identical procedure was performed by treating lipoamide dehydrogenase with pyridoxine-5’-phosphate. Aminocarboxymethylation followed the published procedure (9) by treating reduced lipoamide dehydrogenase (EHz) with iodoacetamide. Cadmium derivative of lipoamide dehydrogenase was prepared by treating the NADH-reduced enzyme with 15-fold CdClz (10). The sensitized photooxidation was carried out as reported (11) by illuminating modified enzyme (45 pg ml-’ in 50 mM phosphate buffer, pH 7.0). Enzyme assays and kinetic studies. Multifunctional activities of lipoamide dehydrogenase and its derivatives were assayed at pH 7.0 in 50 mM phosphate buffer at 25°C (12). The reductase activity followed a decrease in the absorption at 340 nm of an assay mixture (1.0 ml) containing 50 PM, NADH, 250 PM lipoamide (containing 1.0 mru EDTA), and 1.0 + 0.5 Mg enzyme. The transhydrogenase activity was assayed spectrophotometrically (395 nm) of a mixture (1.0 ml) containing 50 pM NADH, 125 pM TNAD+, and 1.0 + 0.5 pg enzyme. The electron transferase activity followed a decrease in the absorption at 420 nm of a reaction mixture (1.0 ml) containing 50 FM NADH, 250 pM K,Fe(CN)G, and 5.0 rt 2.5 pg enzyme. An assay mixture (1.0 ml) of the diaphorase reaction contained 50 pM NADH, 20 pM DCIP (dichlorophenol indophenol), and 5.0 IfI 2.5 pg enzyme and a change in the absorption at 600 nm was followed. Kinetic studies of electron transferase reactions were carried out as described (5) by following rates of a decrease in absorption at 420 nm spectrophotometrically by means of a Perkin-Elmer spectrophotometer (Coleman Model 124) or a Beckman Model 25 spectrophotometer. The reaction mixture (1.0 ml) contained 0.010 to 0.20 mM of NADH, 0.050 to 0.10 mM of K8Fe(CNk and 10-106 nM of lipoamide dehydrogenase of its derivative in 50 mM phosphate buffer, pH 7.0. For pH-dependent studies, 50 mM acetate, phosphate, and tris buffers of appropriate pH values were used. Initial rates in the asymptotic region were fitted to: ?I=

for converging

VAB AB

+ KbA

reciprocal

+ K,B plots

+ Ki.K:, or

PI

556

TSAI VAB v = AB + &A

ET

RESULTS

The one-electron transfer reaction catalyzed by lipoamide dehydrogenase was studied by following rates of NADH consumption at 340 nm which had been corrected for ferri/ferrocyanide absorptions and Fe(CN)g- consumption at 420 nm, respectively. Table I shows that the stoichiometry of [NADH]/[Fe(CN)&] = 0.5 for various NADH to enzyme ratios in the region ([NADH]/[E] > 200) of rate studies.

STOICHIOMETRY CATALYZED

I

OF ELECTRON BY LIPOAMIDE

lNADWlE1 100 200 400 500 750 1000

TRANSFER REACTION DEHYDROGENASE

[NADH]/[Fe(CN)g-] 1.2 0.5 0.6 0.6 0.5 0.4 0.5

+ -t f f f + +

0.3 (aerobic) 0.2 (anaerobic) 0.1 0.1 0.1 0.2 0.2

Note. The electron transfer reaction was carried out in 50 mre phosphate buffer, pH 7.0, at 25°C. The stoichiometry of substrate consumption ([NADHY [Fe(CN):-) was calculated according to: [NADH]/[Fe(CN)z-]

= VW 6::;

TABLE

PI

+ K,B

for parallel reciprocal plots. V, K., Kb, and Ki. are maximum velocity, Michaelis constant for NADH, Michaelis constant for K,Fe(CN),, and inhibition constant for NADH, respectively. Anu&ical methods Ultraviolet-visible spectra were recorded with a Cary 14 spectrophotometer. pH values were measured and adjusted by means of a radiometer, PHM62/TIT60. Aqueous dissolved oxygen concentrations were determined with a YSI Model 57 dissolved-oxygen meter equipped with a Honeywell Electronik 194 recorder.

TABLE

AL.

‘,) #al

where VW and VI&,, were rates of decreases in absorptions at 340 nm (c = 6.22 X 109 M-I cm-’ for NADH) which was corrected for AC (540 M-’ cm-i for Fe(CN):- Fe(CN):and 420 nm (t = 1.06 X 10r M-’ cm-’ for Fe(CN):-), respectively.

II

PROMOTION OF ELECZRON TRANSFER REACTION OF LIPOAMIDE DEHYDROGENASE % Control Riboflavin (10 PM) added Reductase Transhydrogenase Electron transferase Diaphorase

activity

E,

Ec.,

Ec.

100 100

0 139

2 16

11 70

109 108

148 138

110 273

144 123

Note. Multifunctional activities were assayed as described in the text. The controls for E,, ECd, and Ea were prepared in parallel experiments (identical manners) without iodoacetamide, Cd&, and C&O,, respectively.

The high [NADH]/[Fe(CN)i-] with lowering [NADH]/[E] was attributable to an intervening NADH oxidase activity which was suppressed under anaerobic condition. In kinetic studies, the bimolecularity of Fe(CN)i- was evidenced from the nonlinear reciprocal plots of u-l versus [Fe(CN)i-1-l (5). Various approaches to promote ETase reaction of lipoamide dehydrogenase were investigated. Added riboflavin slightly facilitated the rate of ETase reaction (Table II). The absorption spectrum taken at the completion of reaction showed that riboflavin was not destroyed. Under the assay condition, riboflavin did not mediate the redox reaction between NADH and/or Fe(CN)i- in the absence of the enzyme. However, riboflavin was readily bleached by NADH in the presence of lipoamide dehydrogenase (Fig. 1). An enhanced ETase activity (Table II) was observed for aminocarboxymethylation which specifically modified the lipoate site of the catalytic disulfide (9), and cadmium derivatization of the disulfide (10). Although modifications of the catalytic disulfide do not always promote the electron transfer reaction, the enhanced ETase activity of E,, and EM implicates that the catalytic disulfide is not essential for the ETase activity. The observation that the cobaltous complex of lipoamide dehydrogenase (EG) displayed decreased RDase and THase (6) was confirmed in this experiment. Significantly,

ONE-ELECTRON

TRANSFER

REACTION

OF

LIPOAMIDE

557

DEHYDROGENASE

A (nm)

FIG. 1. Difference spectra (2-4) of reduction of lipoamide dehydrogenase by NADH in the presence of riboflavin. The absorption spectrum of 3.8 X 10-s mol of lipoamide dehydrogenase (in 2.50 ml 10 mM phosphate buffer, pH 7.0) in the presence of 3.7 X lo-* mol riboflavin was balanced against the equimoles of enzyme (in the same buffer) (spectrum 2). Both the sample and reference solutions were titrated with 12.5 X lo-* mol of NADH (spectrum 3) and 25.0 X lo-* mol of NADH (spectrum 4). All spectra were taken with a Cary 14 spectrophotometer at 25°C. For a comparison, the spectra of oxidized enzyme (spectrum 1) and reduced enzyme (spectrum 5) are included.

however, ETase activity was increased by 44 f 22% based on the initial rate assay. Kinetic studies of Eco showed that the observed increase in the ETase activity of Eco resulted from decreased Michaelis (K, and Kb) and inhibition (Ki,) constants (Table III). The indeterminately small Ki, was implicated by a family of nearly parallel lines in the reciprocal plots (Fig. 2), since Eq. [l] became Eq. [2] if A % Ki,. The one-electron transfer reaction catalyzed by flavoenzymes proceeds via a semiquinone radical intermediate (2). The neutral radical is favored by a base or an anionic residue whereas the anionic radical is stabilized by a cationic residue of apoenzymes (4, 13). The possible role of amino and carboxyl groups of lipoamide dehydrogenase in the ETase reaction was investigated. No significant change in the ETase activity was observed for lipoamide dehydrogenase treated with methyl acetimidate and methyl benzimidate which amidinated 15 + 4 lysine residues per FAD (8). Pyridoxine-5’-phosphate which reacted with neither amino groups nor carboxyl groups did not affect the multifunctional activity of lipoamide dehydrogenase. Pyridoxal-5’-phosphate which specifically derivatized one lysine residue per FAD (8)

did not affect the ETase activity. However, pyridoxamine-5’-phosphate which formed an amide derivative with carboxyl groups greatly enhanced the ETase activity (Table IV). Illumination of both phosphopyridoxalated (EPLP) and phosphopyridoxamidated (Ernr) enzymes inactivated the RDase activity. Fig. 3 traces the absorption spectrum of Ernr. From the absorption maximum characteristic of the pyridoxamino group at 325 nm (E = 8300 M-’ cm-‘), 2.8 PNP groups per FAD were incorporated. The carboxyl groups of proteins are TABLE

III

KINETIC PARAMETERS FOR ELECTRON TRANSFERASE REACTION CATALYZED BY Co COMPLEX OF LIPOAMIDE DEHYDROGENASE AT pH 7.0,25”C

v (PM mid) K. (PM) & (PM) Kia (PM) V/E, (min-‘)

Control

EC3

160 243 748 73.8

55 93.0 180 -a

3.2

x

103

Note. Kinetic studies were performed nM of the control enzyme and Eo,. a Indeterminately small value (near

1.1 x lo3 by using zero)

50

for Kia.

558

TSAI

ET

AL.

25

350

450 WAVELENGTH (nm)

550

FIG. 3. Absorption spectrum of pyridoxamidated lipoamide dehydrogenase, pH 7.0. 20

40

[NAOH-‘1

FIG. 2. Kinetic action catalyzed

60

60

100

( ~M-I)

studies of electron transferase reby Ec. (50 nM) at pH 7.0 and 25°C.

readily amidated by amines in the presence of carbodiimide (7). Figure 4 illustrates progress curves of amidation with amines bearing different charged groups. Ethylene diamine caused the greatest enhancement in the ETase activity. 2-Aminoethyl sulfonic acid slightly activated the ETase due primarily to the effect of EDC which, as the control reagent, parallel that of 2-aminoethyl sulfonic acid. Propylamine exhibited an effect intermediate between the two charged amines. Table V shows the specific amidation effect on the ETase activity with little variation in the other multifunctional activities. No crosslinkage occurred with ethylene diamine as examined by a sodium dodecyl TABLE MULTIFUNCTIONAL

sulfate-gel electrophoresis. In a double modification experiment, PNP was not incorporated into the aminoethylamidated enzyme (En&. Figure 5 shows reciprocal rate plots of the electron transfer reaction catalyzed by ENd. The increased ETase activity of ENa is associated with the increased catalytic efficiency as well as decreased Michaelis constants for both NADH and Fe(CN)i-(TableVI).pH-Dependentstudies of the ETase reactions were hindered by the competing oxidase activity at low pH and a removal of FAD at high pH. Within a narrow pH range studied, however, significant differences between the native enzyme and ENHt in pH profiles for V/E, and Ki, were observed (Fig. 6). The pH-dependent V/E, for the native enzyme was bellshaped while it was sigmoid for ENHi. Furthermore (Kia)-’ for the native enzyme dropped at high pH whereas that of ENa was independent of pH. IV

ACTIVITIES OF PHOSPHOPYRIDOXAL AND PHOSPHOPYRIDOXAMIDATED DEHYDROGENASES AND EFFECTS OF PHOTOOXIDATION % Control E PLP

Reductase Transhydrogenase Electron transferase Diaphorase Note. The subscript hv denotes (45 pg ml-’ in 50 rnM phosphate

125 106 95 145

%m 37 88 118 134

EPNP

65 75 670 215

LIPOAYIDE

activity GNP/~,

24 65 250 430

illumination with a 200-W photo-flood lamp 25 cm from buffer at pH 7.0) isothermally at 25°C for 30-45 min.

EPP

98 110 94 112 an enzyme

E PPh. 92 106 101 123 solution

ONE-ELECTRON

6-

REACTION

TRANSFER

OF

LIPOAMIDE

559

DEHYDROGENASE

/ ,/”

4-

,0--o-

4 ,/

2 -0’ ,o

I

I

1

20 40 REACTION

I

69 TIME

60

l ‘0

I 100

(mm)

FIG. 4. Changes in electron transferase activity of lipoamide dehydrogenase during amidation. Amidation (amide formation) was carried with ethylene diamine (0), propylamine (O), or 2-aminoethylsulfonic acid (taurine, A) as the nucleophile effected by EDC at pH 5.5 DISCUSSION

Lipoamide dehydrogenase as a component enzyme of 2-0~0 acid dehydrogenase complexes mediates the redox reaction of protein-bound lipoamide physiologically (14). In its free form, the enzyme catalyzes reversible NADH-linked reduction of lipoamide via two-electron transfer (3, 4). However, one-electron transfer termed electron transferase (ETase) reaction can be forced to proceed, though inefficiently, by the use of one-electron acceptors, such as Fe(CN)i-. The one-electron transfer reaction is believed to proceed via flavoseTABLE MULTIFUNCTIONAL LIPOAMIDE

V

80 75 680 145

88 96 313 116

studies by EM

of electron transferase re(15 nM) at pH 7.5, 25°C.

miquinone radical as the intermediate (2, 13). Therefore, the ETase reaction of lipoamide dehydrogenase can be promoted by various approaches which stabilize the flavosemiquinone radical. It is well recognized that the reduced and oxidized flavins form the flavoquinhydrone complex which dismutates (15-17). Furthermore, the presence of polymers (18) or the immobilization of riboflavin (19) greatly enhances the rate of riboflavin reduction by NADH. Since the addition of

ACITVITIES OF AWDATED DEHYDROGENASES % Control

Reductase Transhydrogenase Electron transferase Diaphorase

FIG. 5. Kinetic action catalyzed

TABLE

VI

KINETIC PARAMETERS FOR ELECIXON TRANSFERASE REACTION CATALYZED BY AMINOEXHYLAMIDATED LIPOAMIDE DEHYDROGENASE

activity

92 102 126 105

Note. The control activities were based on 196% for controls in the presence of respective amines. The control in the presence of EDC gave 91% (RDase), 85% (THase), 136% (ETase), and 110% (DPase) with respect to the amine control. The amidation was carried out at pH 5.5 for 75 + 15 min when the maximum activity was reached. The multifunctional activities were assayed as described.

Control V (PM mini) Ka (PM) Kb (PM) Kia (PM) V/E, (mini)

296 210 621 50.7 2.96

+ k + f X

EN&

20.1 15.9 102 3.5 lo3

152 26.1 154 39.2 7.60

2 23.6 k 6.3 + 36.8 f 9.7 x 103

Note. Kinetic studies were performed by using 196 and 20 mM (normalized) of the control enzyme and EM, respectively, at pH 7.5,25”C. Average values of duplicate determinations from three preparations are given.

560

TSAI

P-*-i4

PH

FIG. 6. pH rate profile for electron transfer reaction catalyzed by native lipoamide dehydrogenase (0) and its ethylene diamine derivative (EN&, 0). The inset shows pH effect on reciprocal Ki,.

riboflavin specifically affects the ETase reaction, the added riboflavin likely dismutates the reduced FAD to facilitate the electron transfer. The active site disulfide of lipoamide dehydrogenase and glutathione reductase is stipulated to destabilize the radical formation in these two enzymes (20). E,, in which the substrate site of the catalytic disulfide is aminocarboxymethylated (9) and ECd in which the disulfide is linked to cadmium (10) indeed display the enhanced ETase activity. In model systems, the flavosemiquinone radical is stabilized by chelation with metal ions (21,22) presumably via O(4a) and N(5) of the isoalloxazine ring (23,24). Cobaltous ion forms a stable complex with reduced lipoamide dehydrogenase at pH 10 (6) resulting in the enhanced ETase activity. The facilitated initial rate of the electron transfer arises from the decreased Michaelis constants (K, and &) and, in particular, inhibition constant (Ki,) which diminishes. This suggests a tight initial binding of NADH to EcO or the formation of a stable enzyme intermediate prior to the binding of Fe(CN)g-. Divalent cations are capable of interacting with the flavosemiquinone which favors the radical chelate formation (25). It is conceivable that the stability of the flavosemiquinone intermediate of Eco may be enhanced by the formation of the radical chelate. Pyridoxamine-5’-phosphate which, in the

ET

AL.

presence of EDC, amidates carboxyl residues of lipoamide dehydrogenase greatly promotes the ETase reaction. The specific suppressive effect of carboxyl groups on the ETase activity of lipoamide dehydrogenase is demonstrated by the ineffectiveness of PLP and pyridoxine$‘-phosphate to enhance the ETase activity. An extensive amino acid sequence homology exists between lipoamide dehydrogenase and its closely related enzyme, glutathione reductase (26). The complete amino acid sequence (27) and X-ray crystallographic structure (28) of glutathione reductase have been elucidated. Three carboxyl residues, Glu-201, Glu-472, and Asp-331 which are paired with Lys-66, the catalytic His472 and Arg-291, respectively, are located within the catalytic center of glutathione reductase (29). In the present study with lipoamide dehydrogenase, approximately three carboxyl residues per FAD are phosphopyridoxamidated. The ability of PNP to sensitize photoinactivation of EpNp indicates the proximity of the amidated carboxy1 residues to the active site histidine. The inability of aminoethylamidated lipoamide dehydrogenase (EN& to incorporate PNP implicates that the common carboxyl groups are amidated. The promotion of ETase reaction by amidation with various amine nucleophiles provides illuminating information. Three isosteric amines, H,N(CH,),NH3+, H2N(CH&CH3, and H2N(CH&SO;, introduce cationic, neutral, and anionic groups onto the anionic carboxyl groups of lipoamide dehydrogenase. The ETase activity of ENmwhich reverses the charge character, increases by six- to sevenfold while that of Eso,, which retains the same ionic character, remains at the control level. The ETase activity of ECHs is intermediate between the two amidated enzymes. Steadystate kinetic studies reveal that the charge reversal of the carboxyl group by the aminoethyl group in ENm facilitates not only the interaction of Fe(CN)i- but also the catalytic efficiency of the transfer reaction. To force the one-electron transfer, the flavin coenzyme of lipoamide dehydrogenase must be reduced to the flavosemiquinone intermediate which may exist in

ONE-ELECTRON

TRANSFER

REACTION

the neutral or anionic radical (2, 13). The former is favored by a juxtaposed anionic residue while the latter is stabilized by a cationic residue of apoenzyme. Attempts to detect the flavosemiquinone radical in lipoamide dehydrogenase have not been successful (20,30). If the increased catalytic efficiency for the one-electron transfer is interpreted in terms of the stabilized flavosemiquinone radical by the cationic aminoethyl group(s) of En@, the participation of an anionic radical is implicated to account for the enhanced ETase activity of ENHi. Since the carboxyl residues in the catalytic center are paired with the cationic residues as inferred from glutathione reductase (29), the aminoethylamidation may alternatively free the paired cationic residues of ENa which, in turn, stabilize the anionic radical. The active site disulfide of lipoamide dehydrogenase and glutathione reductase tends to destabilize the radical formation in these two enzymes. It is likely that the juxtaposed carboxyl group(s) may further suppress the formation of the anionic radical, thereby inhibit the ETase activity. The release of this suppression moderately increases the ETase activity of the propylamidated enzyme (EC&, The stabilizing effect of the cationic aminoethyl group further promotes the ETase reaction catalyzed by the aminoethyl amidated enzyme (ENH$. ACKNOWLEDGMENTS This work was supported by a grant from the Natural Science and Engineering Research Council of Canada. D.M.T. is a holder of NSERC graduate scholarship and A.J.W. is a holder of Ontario graduate scholarship.

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