©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Active Site Studies of DT-diaphorase Employing Artificial Flavins (*)

(Received for publication, May 17, 1994; and in revised form, December 1, 1994)

Gabriella Tedeschi (1) Shiuan Chen (2) Vincent Massey (1)(§)

From the  (1)Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0606 and the (2)Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is an FAD-containing enzyme that catalyzes the 2-electron reduction of quinones to hydroquinones using either NADH or NADPH as the electron donor. In this study, FAD was removed by dialyzing the holoprotein against 2 M KBr, and synthetic analogs of FAD were substituted in the flavin binding site as structural probes. Spectral analysis indicates that the benzoquinoid forms of 8-mercapto-FAD and 6-mercapto-FAD are stabilized on binding to the enzyme. This is consistent with the fact that the native flavoprotein forms the anion flavin radical upon photoreduction and suggests the presence of a positive charge near the N(1)C(2)O position of the isoalloxazine ring. Reactivity studies using 8-chloro- and 8-mercapto-flavins suggest that the 8 position of the FAD is accessible to the solvent. However, the rates of the reactions were dramatically decreased in the presence of the competitive inhibitor, dicumarol. 6-Mercapto-, 6-thiocyanato-, 6-azido-, and 6-amino-flavins were also used as structural probes. The results indicate that the 6 position is accessible to solvent. Dicumarol binding increases the pK of the enzyme-bound 6-mercapto-flavin from below pH 5.0 to higher than pH 9.0. The results suggest that DT-diaphorase shows the same properties as the C-C transhydrogenases, and the binding of dicumarol elicits a conformational change or an adjustment in the polarity of the FAD pocket. The enzyme reconstituted with oxidized 5-deaza-FAD has significant catalytic activity, confirming that DT-diaphorase is an obligatory 2-electron transfer enzyme and plays a role in the detoxification of quinones and quinoid compounds by reducing them to the relatively stable hydroquinones.


INTRODUCTION

NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is a cytosolic FAD-containing protein that catalyzes the reduction of quinones to hydroquinones using either NADH or NADPH as the electron donor(1) . Besides quinones, DT-diaphorase reduces nitro compounds(2, 3) , azo-dyes(4) , and hexavalent chromium compounds (5) as well as ferricyanide. The rat liver enzyme expressed in Escherichia coli is a dimeric molecule containing one non-covalently bound FAD per protein monomer of 30 kDa(6) . According to Iyanagi and Yamazaki (7) the oxidative half-reaction is a 2-electron transfer process without formation of the reactive semiquinone intermediate. Therefore, DT-diaphorase is thought to function in the cellular detoxification of quinones by shunting these compounds away from the free radical state. This is presumed to result in a decrease of oxygen free radicals, which are generated by redox cycling of semiquinones in the presence of oxygen(8) . In addition, the enzyme seems to be involved in the reductive activation of the cytotoxic anti-tumor quinones (important drugs for cancer chemotherapy(9) ), and it can function as one of the vitamin K reductases in the blood coagulation process(10) . While the physiological roles of the enzyme have been extensively studied, at present the structure-function relationships are not clearly understood. Analysis of the primary structure revealed that the enzyme does not share significant sequence homology with other flavoproteins (11, 12) . Chemical modification and site-directed mutagenesis studies suggest that DT-diaphorase possesses different binding sites for NAD(P)H, quinones, and the FAD prosthetic group(6, 13, 14, 15, 16, 17) . In particular, Ma et al.(18) proposed that the isoalloxazine moiety of the flavin is located at a site close to the NAD(P)H domain, and a beta-turn-alpha structure is involved in FAD binding. Some information is available regarding the binding pocket of nicotinamide and the competitive inhibitor dicumarol, but the topography of the FAD domain is not well characterized. In this study, the flavin cofactor was removed, and synthetic analogs of FAD were substituted as structural probes, providing new insights into the environment of the flavin binding site and into the interaction between dicumarol and the enzyme.


MATERIALS AND METHODS

DT-diaphorase overexpressed in E. coli was purified by Paulis Deng according to a previously reported procedure(6) . NADH, NADPH, NADP, and dicumarol were from Sigma. 8-Mercapto-FAD was prepared from 8-chloro-FAD by reaction with sodium sulfide(19) . 6-Mercapto-FAD was prepared from 6-thiocyanato-FAD by reaction with 1,4-dithiothreitol(20) . The apoenzyme was prepared by dialyzing the holoenzyme for 3 days at 4 °C against 200 mM potassium phosphate, pH 6.0, containing 0.3 mM EDTA, 20% glycerol, 2 M KBr, and activated charcoal (about 1 g added to 200 ml of dialyzing fluid) to adsorb FAD as soon as it passed through the dialysis membrane. When the yellow color of the enzyme was no longer visible, the dialysis medium was changed to 200 mM potassium phosphate, pH 7.0, containing 0.3 mM EDTA and 20% glycerol. Reconstitution with oxidized flavin analogs was performed in the same buffer at ice temperature with variable amounts of apoprotein and excess of flavin. Unbound flavin was removed by repeated ultrafiltration on a CM30 microconcentrator (Amicon). The spectrum of the filtrate was recorded and subtracted from that of the mixture before ultrafiltration to yield the spectrum of the reconstituted enzyme. Dissociation constants were then calculated based on the concentrations of the starting apoenzyme and the concentration of the flavin in the filtrate.

DT-diaphorase assays were carried out at 25 °C in the reconstitution buffer by following the decrease in absorbance at 340 nm in the presence of an excess of FAD or FAD analogue. NADPH was used as electron donor, and 2-hydroxy-1,4-naphthoquinone was used as electron acceptor. The activity of the apoenzyme due to residual FAD (1-5% initial) was always measured and subtracted from the measured activity of reconstituted enzyme. In all cases the double-reciprocal plots present a set of parallel lines indicating a ping-pong mechanism as reported for the native enzyme (21) but without definable values of V(max) and K for electron donor or acceptor, as described for other acceptors(22) .

Anaerobic experiments were performed in all glass apparatus (23) by sequential evacuation and re-equilibration with oxygen-free argon.

Photochemical reduction experiments were performed by irradiating the solution with a Sun Gun (Smith Victoria Corp., Griffith, IN). Visible spectra were taken using a Hewlett Packard 8452 A diode array spectrophotometer or a Varian Cary 3 UV-visible spectrophotometer. If not otherwise indicated, all the reactions were carried out at 25 °C in 0.2 M potassium phosphate, pH 7.0, containing 0.3 mM EDTA and 20% glycerol.


RESULTS AND DISCUSSION

Preparation, Properties, and Reconstitution of DT-diaphorase Apoprotein

DT-diaphorase apoprotein was obtained by dialyzing the holoenzyme against 2 M KBr and activated charcoal. The addition of glycerol and the use of phosphate buffer at high ionic strength were essential to avoid protein denaturation and allowed reactivation of the apoprotein. The yield of apoenzyme in terms of protein recovery was about 60-70%, and the enzyme preparation regained 80% of its specific activity with respect to the native holoenzyme after incubation on ice with excess of FAD. The activity recovery was independent of the incubation time. The apoprotein was stable for 1-2 months if stored at -20 °C, and the spectral properties of the FAD-reconstituted enzyme were virtually identical with those of the native protein(4) . The FAD analogs used in this work bind to the apoenzyme with dissociation constants approx10M (Table 1). However, replacement of the flavin N(5) by carbon (5-deaza-FAD) results in a very large decrease in binding affinity (K(d) 1.4 times 10M at 25 °C compared with 1.7 times 10M for 1-deaza-FAD). Most flavoproteins bind 1- and 5-deazaflavin analogs with almost equal affinity(24, 25) . It is therefore likely that in the case of DT-diaphorase, the N(5) position of the flavin is involved in hydrogen bond interaction with the protein moiety.



All the analogs are enzymatically active and can be reduced by NAD(P)H under anaerobic conditions. Using 2-hydroxy-1,4-naphthoquinone as electron acceptor, there are no definable values of k and K(m), as already described for the native enzyme(22) . The second order rate constants for the reaction of the reduced flavoprotein with the quinone (k(2)) are collected in Table 1along with the E(m)(7) values of the flavin analogs. Similar to results with the native enzyme, with all the FAD analogs the reduction of the flavin appears to be much faster than the reoxidation, since the observed steady-state rates were very little influenced by the concentration of NADPH(22) .

Accessibility of the 8 Position to Solvent

Reconstitution of DT-diaphorase with 8-chloro-FAD yields a nonresolved spectrum closely similar to that of the native enzyme (Fig. 1). The result rules out the possibility of a covalent bond between the protein and the flavin by displacement of the 8-chloro group as found in lipoyl dehydrogenase(28) .


Figure 1: Reaction of 8-chloro-FAD-DT-diaphorase with thiophenol. The reconstituted enzyme before (1) and after (2) 50 µM thiophenol at pH 6.0 and 25 °C is shown. Inset, plot of apparent rates versus thiophenol concentration. The rate of the reaction was calculated by following the change in absorbance at 490 nm.



Accessibility of the flavin 8 position was explored by reacting the reconstituted enzyme with sulfur nucleophiles. Before the reaction, the reconstituted enzyme was passed through a Sephadex G-25 column equilibrated in 0.2 M phosphate buffer, pH 7.0, 0.3 mM EDTA, and 20% glycerol to remove excess of free flavin. Fig. 1shows the spectral changes encountered on the addition of 50 µM thiophenol to 8-chloro-FAD-DT-diaphorase. The typical two-banded spectrum is replaced by an intense single maximum at 480 nm with a shoulder at 460 nm. This spectrum is clearly different from that of free 8-phenylmercapto-FAD(29) , suggesting that the protein can accommodate this somewhat bulky substituent without release of the flavin. Solvent accessibility has also been examined under the same conditions as in Fig. 1but with 1,4-dithiothreitol, sodium sulfide, and beta-mercaptoethanol (data not shown) used as nucleophiles. As shown in Table 2, it is clear that the 8-chloro group of 8-chloro-DT-diaphorase reacts faster than does the 8-chloro group of the free flavin, suggesting that this position is solvent-exposed and that the protein can stabilize the tetrahedral intermediates of the reactions(32) . Monophasic kinetics were observed with all thiol reagents, and simple second order rate constants were calculated in each case. These results confirm that the reconstituted enzyme is a homogeneous species and that no free flavins are present.



In particular, sodium sulfide at pH 5 and pH 8 effects the displacement of chloride, giving a spectrum typical of the benzoquinoid form of 8-mercapto-FAD(19) . A titration of free 8-mercapto-FAD with apoenzyme results in a non-resolved spectrum with an extinction coefficient of 30,200 M cm at 590 nm (Fig. 2). In this form, the negative charge is carried over the N(1)C(2)O locus rather than on the benzene subnucleus of the flavin, and its stabilization by the enzyme suggests the presence of a positive charge in the FAD binding site of DT-diaphorase. As expected, the decreased nucleophilicity of this form results in much lower reactivity of the enzyme-bound-8-mercapto-FAD toward thiol reagents. The second order rate constants for iodoacetamide and methyl methanethiolsulfonate reactions were 1.07 M min and 342 M min, respectively (data not shown), much lower than the reaction with free 8-mercapto-FAD under the same conditions(32) .


Figure 2: Titration of 8-mercapto-FAD with apo-DT-diaphorase. 8-Mercapto-FAD (3.4 µM), curve 1, was titrated with apoenzyme 0.77 (2), 1.73 (3), and 3.4 µM (4) at pH 8.0. Inset, change in extinction coefficient at 520 (bullet) and 590 () nm on addition of apoenzyme.



By reacting 8-mercapto-FAD-DT-diaphorase with hydrogen peroxide, we further confirmed the accessibility of the 8 position to solvent. The oxidation occurs in two distinct steps. The extinction at 590 nm dropped fairly rapidly to 16,000 M cm, and the maximum was shifted to 620 nm, suggesting the presence of a deprotonated thiol oxide, as previously observed for 6-mercapto-FAD reacted with hydrogen peroxide (33) and for 8-mercapto-FAD-NADH peroxidase after peroxide addition(34) . The second order rate constant for the initial oxidation of the flavin thiol was 37.5 M min (Fig. 3A). In the second step of the reaction, the flavin-8-sulfenic acid undergoes further oxidation to either the deprotonated sulfinic or sulfonic acid (without further detectable spectral change between the latter) and with a second order rate constant of 3.5 M min (Fig. 3B). Reactivity of the flavin thiol with m-chloroperbenzoic acid is also high and produces spectral changes similar to those found on addition of hydrogen peroxide (data not shown).


Figure 3: Spectral course of the reaction of 8-mercapto-FAD-DT-diaphorase with 3.45 mM hydrogen peroxide. 1, starting spectrum; 2 and 3, spectra of S-oxide derivative; 4, spectrum of sulfinate/sulfonate derivative. Spectra in panelA were recorded before (curve1) and 2, 5, and 9 min after addition of H(2)O(2). Spectra in panelB were recorded at 12, 18, 25, 32, 42, and 89 min after H(2)O(2). The rates of the reaction were calculated from the decrease in absorbance at 600 nm.



Addition of an excess of 1,4-dithiothreitol immediately upon completion of the first reaction step or after the final oxidation results in a displacement of S-oxide or SO(2)=/SO(3)=groups and the formation of a 1,4-dithiothreitol derivative (spectrum similar to that shown in Fig. 1), suggesting that the protein can stabilize the tetrahedral intermediate of this reaction. This observation for 8-substituted flavin establishes a major difference from 6-SO-flavins that can be reduced by 1,4-dithiothreitol and 6-SO(2)=/SO(3)= flavins that do not react with 1,4-dithiothreitol(33) . The stability of the flavin 8-S-oxide derivative was also tested under anaerobic conditions in the presence of catalase to avoid further oxidation. As expected, no further spectral changes were detected after the addition of catalase, indicating the absence of any nucleophilic groups in the vicinity of the flavin 8 position that can displace the S-oxide group.

The same conclusions can be reached using 8-OH-FAD as spectral probe at different pH values (pH 4, 5.5, and 10). DT-diaphorase at pH 5.5 stabilizes the anionic form of 8-hydroxy-FAD with maximum at 480 nm (data not shown). This result was taken as a further evidence for the existence of a positive charge near the N(1)C(2)O position and the absence of a negative charge near the 8 position of the flavin. The binding with apoprotein, in fact, does not result in an increase in the pK(a) of the 8-OH-FAD enzyme (pK(a) = 4.8 for the free flavin) as observed, for example, in Old Yellow Enzyme (35) .

Several of the properties of flavoprotein oxidases as a class have been attributed to the presence of such a positively charged group in the FAD binding site(36) . Since among these are the ability to form an N(5) adduct with sulfite, the reactivity of native enzyme and 8-mercapto-FAD with this reagent was also examined. No spectral perturbations were observed by reacting normal FAD-DT-diaphorase with 10 mM sodium sulfite, indicating no adduct formation at the flavin N(5) position.

As expected, the reaction of 8-mercapto-FAD-DT-diaphorase (wavelength maximum 590 nm, = 30,200 M cm) with sulfite yields 8-sulfonyl-FAD enzyme (wavelength maximum 460 nm, = 9200 M cm), and no further reaction occurs at the N(5) position (37) (data not shown). A negatively charged protein residue near the N(5) position, repelling approach to the sulfite ion, could be responsible for such failure, as well as the presence of a hydrogen bond acceptor near the N(1) locus of the flavin instead of a hydrogen bond donor. There is generally a close correlation among the properties of the 8-mercapto-flavoprotein and the properties of the semiquinoid form of the native enzyme(19) . In particular, the 8-mercapto enzyme is easily reduced by substrate for those flavoproteins, which in the native state give little or no thermodynamic stabilization of a semiquinoid species. In the case of DT-diaphorase, the 8-mercapto-FAD enzyme can be reduced by 70 µM NADH under anaerobic conditions and can be slowly reoxidized by oxygen without any evidence for a semiquinone intermediate. The same result was obtained for the native FAD enzyme. This is consistent with the fact that the native flavoprotein thermodynamically stabilizes only 8-10% of the red anionic semiquinone, with maxima at 382 and 452 nm, upon photoreduction in the presence of EDTA, 5-deazaflavin-3-sulfonate, and benzylviologen as redox mediator(22) .

Accessibility of the 6 Position to Solvent

6-Azido-, 6-amino-, 6-mercapto-, and 6-thiocyanato-flavins were used as structural probes for the 6 position (20) . 6-Azido-FAD binds to the apoprotein with a K(d) of 3.5 times 10M and an extinction coefficient of 17,500 M cm at 429 nm. Light irradiation of 6-azido-FAD enzyme yields easily a derivative identified as 6-amino-FAD because its spectral properties were identical to those of 6amino-FAD-DT-diaphorase obtained after reduction of 6-azido-FAD-DT-diaphorase by NADPH (extinction coefficient, 17,300 M cm at 434 nm). The product is not fluorescent, indicating that it is still bound to the enzyme but without covalent fixation of the flavin to the protein, since the flavin is removed quantitatively by denaturation of the protein with 5% trichloroacetic acid or by heat treatment. These results are indicative of the flavin 6 position being exposed to solvent.

6-Azido-FAD-DT-diaphorase is also converted to the 6-amino-FAD enzyme by aerobic turnover with NADPH. Within seconds of the addition of a substoichiometric concentration of NADP and an NADPH-generating system (glucose-6-phosphate and glucose-6-phosphate dehydrogenase) to an anaerobic solution of 6-azido-FAD enzyme, there was substantial bleaching of the 429-nm absorption, followed by the appearance of the 6-amino-FAD spectrum after opening to air. This product can be also reduced by NADPH under anaerobic conditions.

6-Thiocyanato-FAD binds to DT-diaphorase with an absorption maximum at 441 nm. The resulting holoprotein reacts very fast with 1,4-dithiothreitol, suggesting also that the 6 position is accessible to solvent. A second order rate constant of 1750 M min was calculated for the reconstituted enzyme in comparison with the value of 1000 M min obtained for the free flavin under the same conditions. As expected for the presence of a positive charge near the N(1)C(2)O position of the flavin, also in this case the anionic form of 6-mercapto-flavin was highly favored, as shown in Fig. 4by the presence of the long wavelength absorbance.


Figure 4: Conversion of 6-thiocyanato-FAD-DT-diaphorase to 6-mercapto-FAD-DT-diaphorase. 6-Thiocyanato-FAD enzyme (1) was reacted with 196 µM 1,4-dithiothreitol at 25 °C, and the time course of the reaction was followed at 441 nm. 2, spectrum of the resulting 6-mercapto-FAD enzyme in the anionic form. Inset, plot of apparent rates versus 1,4-dithiothreitol concentration in the absence (circle) and in the presence (times) of 30 µM dicumarol.



In keeping with the benzene ring of the bound 6-mercapto-flavin being readily accessible to solvent, reaction with methyl methanethiolsulfonate is very fast, and the 6-mercapto-FAD spectrum can be regenerated by the addition of 1,4-dithiothreitol (data not shown). 6-Mercapto-FAD enzyme reacts also with iodoacetamide with a second order rate constant of 85 M min (150 M min for the free flavin under the same conditions) and with m-chloroperbenzoic acid or hydrogen peroxide to produce the flavin-6-S-oxide (180 M min for hydrogen peroxide). As observed with lactate monooxygenase(33) , this species is very resistant to further oxidation, and extended incubation with excess of peracid is required for sulfinate/sulfonate formation. It is probably the anion form of the S-oxide that is stabilized because of a positively charged protein residue in the vicinity of the flavin N(1) position, which stabilizes anionic flavin species, including the 6-mercapto flavin(33) .

Taken together, the preceding results describe a flavin binding pocket that is accessible to solvent along one entire edge of the isoalloxazine ring system, from position 8 to position 6. This feature seems to be common to all the pyridine nucleotide-dependent flavoproteins so far studied(36) . Furthermore, as indicated by the non-resolved spectrum, the general flavin environment is hydrophilic, and positively charged protein residue(s) exist near the N(1)C(2) locus. The enzyme does not react with sodium sulfite and forms a red semiquinone that is not thermodynamically stabilized. This description, although it constitutes a minimal picture of the flavin-protein interactions, suggests that DT-diaphorase shows the same properties as the C-C transhydrogenases according to the general classification proposed by Massey and Hemmerich (38) for flavoenzymes.

1-Deaza-FAD-DT-diaphorase

On binding the apoenzyme to 1-deaza-FAD, we obtained a non-resolved spectrum with a maximum at 540 nm. The reconstituted enzyme does not stabilize thermodynamically a semiquinone form after photoreduction in the presence of EDTA, benzylviologen, and 5-deazaflavin-3-sulfonate, similar to the results obtained for the native enzyme.

1-Deaza-FAD-DT-diaphorase was also reduced by NADPH and reoxidized by oxygen or diazaquinone as electron acceptor without any evidence for semiquinone formation.

Use of 5-Deaza-FAD as Mechanism Probe

In 1970, Iyanagi and Yamazaki (7) demonstrated that DT-diaphorase was the only apparent example of a quinone reductase in which the quinone acceptor is reduced without formation of quinone free radicals. It was proposed that if the 1-electron reduced semiquinone were formed as an intermediate in the oxidative half-reaction, it may not be easily removed from the active site and would be reduced to its hydroquinone form by a successive 1-electron transfer reaction. We checked this hypothesis using 5-deaza-FAD as a probe, since semiquinone formation with this flavin is energetically highly unfavorable, thus precluding any role of 5-deaza-flavin radicals in 1-electron catalysis(31) . The FAD analogue is thus a good probe for the differentiation between 1- and 2-electron processes. 5-Deaza-DT-diaphorase is enzymatically active. The specific activity was about 5% of that of the native enzyme using 2-hydroxy-1,4-naphthoquinone as substrate (Table 1). In contrast, no activity was observed with the obligatory 1-electron acceptor ferricyanide. Our results confirm that DT-diaphorase can react as an obligatory 2-electron donor, and the flavin radical is not a catalytic intermediate. The result is also consistent with the observation that the enzyme does not stabilize a blue neutral flavin radical like many dehydrogenases that are involved in obligatory 1-electron transfer reactions(36) . This unique feature of DT-diaphorase provides a conceivable explanation for the indications that the enzyme plays a role in the detoxication of quinones by reducing them to the relatively stable hydroquinones before they react covalently with macromolecules (arylation) or become involved in redox cycles(8) .

Dicumarol Binding to the Reconstituted Enzyme

A striking feature of DT-diaphorase is its high sensitivity to dicumarol. This potent anticoagulant binds to the oxidized form of the enzyme competitively versus the reduced pyridine nucleotide and non-competitively versus electron acceptors. Binding affects the spectral properties of the enzyme-bound flavin by causing a decrease in absorbance at 317, 383, and 452 nm(4) . By site-directed mutagenesis studies, Ma et al.(18) proposed that the binding site for dicumarol may be close or overlap with the binding pocket for NAD(P)H and for FAD. Therefore, it was interesting to analyze the properties of reconstituted enzymes in the presence of dicumarol. Spectral changes very similar to those observed with the native protein were obtained after addition of 10-30 µM dicumarol to the enzyme reconstituted with all the FAD analogs, the only exception being 8-mercapto-FAD, possibly indicating lack of binding of dicumarol to this form of the enzyme. The rates of the reaction between 8-mercapto-FAD enzyme and methyl methanethiolsulfonate, iodoacetamide, or hydrogen peroxide were not affected by the presence of dicumarol. However, this competitive inhibitor dramatically decreased the rate of the reaction of 8-chloro-FAD enzyme with thiophenol and 1,4-dithiothreitol (2 and 1.5%, respectively, in comparison with the rates calculated in the absence of dicumarol), suggesting that the 8 position is still solvent-accessible but the protein does not stabilize any more the tetrahedral intermediate involved in the nucleophilic displacement of chloride by thiols.

An interesting observation was made using 6-substituted FAD analogs. In this case, dicumarol binding had no effect on the rates of the reactions described in a previous section but increased the pK(a) of the enzyme-bound 6-mercapto-FAD from below pH 5.0 to greater than pH 9.0. In Fig. 5, the spectrum of the anionic form of 6-mercapto-FAD enzyme is shown and the neutral form obtained after adding dicumarol at pH 7.0. At this pH, the free flavin is largely ionized (pK(a) = 5.9(20) ) with the thiol group bearing the negative charge. The negative charge is shifted mainly to the N(1) locus after binding to the protein, as indicated by the absorbance at long wavelengths (Fig. 5, inset). The same effects of dicumarol were obtained from pH 5.5 to 8.5. Since the pK(a) for dicumarol free or bound to the protein is 7.5(39) , the increase in the pK(a) of the enzyme-bound 6-mercapto-FAD is not related to the ionization state of the dicumarol. Possibly, binding of the competitive inhibitor elicits a conformational change or an adjustment in the polarity of the FAD binding site favoring such phenomena. Whatever the mechanism, the ineffectiveness of dicumarol on the spectral properties of 8-mercapto-FAD at pH 5-8 suggests that the positive charge near the N(1)C(2)O position of the flavin is not affected, although the environment of the 8 position is changed since the rates of nucleophilic substitution reactions are very much reduced.


Figure 5: Dicumarol effect on 6-mercapto-FAD-DT-diaphorase. Spectra of 6-mercapto-FAD-DT-diaphorase in the absence (1) and in the presence (2) of 37 µM dicumarol at pH 7.0. The same result was obtained in 5 mM sodium pyrophosphate buffer, pH 8.5, and 0.1 M sodium acetate, pH 5.0. Inset, titration of 6-mercapto-FAD with apo-DT-diaphorase. 9.3 µM 6-mercapto-FAD (1) was titrated with 1.7 (2) and 9.3 µM (3) apoenzyme at pH 8.5. Spectra are not corrected for dilution.



In distinction to the results with dicumarol, the pK(a) of 6-mercapto-FAD enzyme and the reactivity of the 8 position do not change in the presence of 40-50 µM NADP or quinones like benzoquinone and menadione, which are known to be very good substrates for DT-diaphorase. These observations could be interpreted as being due to different binding sites for dicumarol and NADPH, despite dicumarol being a competitive inhibitor against NADPH in steady-state assays(4) . However, no kinetic evidence could be obtained for binding of either pyridine nucleotides or quinones, suggesting that the binding affinity of the enzyme for these compounds is at best very weak(22) . For this reason, lack of effect of pyridine nucleotides and quinones on various properties of the enzyme are difficult to interpret.


FOOTNOTES

*
This work was supported by Grant GM-11106 from the U. S. Public Health Service and Grant 963 GI from the American Heart Association, Greater Los Angeles Affiliate (to S. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.


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