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A note on the inhibition of DT-diaphorase by dicoumarol
Free Radical Biology & Medicine, Vol. 11, pp. 77-80, 1991 Printed in the USA. All fights reserved.
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0891-5849/91 $3.00 + .00 Copyright ¢~1991 PergamonPress plc
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Brief Communication A NOTE ON THE INHIBITION OF DT-DIAPHORASE BY DICOUMAROL PETER C. PREUSCH,* DAVID SIEGEL,t NEIL W . GIBSON,~ a n d DAVID Rosst *Department of Chemistry, University of Akron, Akron, OH 44303. Present address: Division of Research Grants, National Institutes of Health, Bethesda, MD 20892; tMolecular Toxicology and Environmental Health Sciences Program, School of Pharmacy, University of Colorado, Boulder, CO 80309; :~Division of Pharmaceutics, School of Pharmacy and Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA (Received 9 January 1991; Accepted 5 February 1991) A b s t r a c t - - T h e participation of DT-diaphorase or NAD(P)H:(quinone acceptor) oxidoreductase (E.C. 1.6.99.2) in metabolism or in events leading to toxicity is often implied on the basis of the inhibitory effects of dicoumarol. DT-diaphorase functions via a ping pong bi-bi kinetic mechanism involving oxidized and reduced flavin forms of the free enzyme. Dicoumarol, a potent (Ki = 10 riM) inhibitor, binds to the oxidized form of the enzyme, competitively versus reduced pyridine nucleotide. Inhibition is effectively complete at 1 p,M dicoumarol in typical studies using DCPIP, one of the best known substrates for the enzyme, as electron acceptor. The antitumor quinone Diaziquone (AZQ) is a poor substrate for DT-diaphorase relative to DCPIP, but effective inhibition of its reduction requires ten-fold higher concentrations of dicoumarol than for inhibition of DCPIP reduction under otherwise similar conditions. The variable inhibition of DT-diaphorase by dicoumarol dependent on the efficiency of the electron acceptor can be explained on the basis of the complete rate equation describing its ping pong type kinetic mechanism. Thus, the concentration of dicoumarol used to inhibit DT-diaphorase must be chosen carefully and consideration should be given to the efficiency of the electron acceptor. The absence of an inhibitory effect using low doses of dicoumarol cannot rule out a reaction mediated by DT-diaphorase. Although higher doses of dicoumarol may be required to inhibit DT-diaphorase mediated metabolism of less efficient electron acceptors, the use of such doses in cells may also affect biochemical processes other than DT-diaphorase and should be approached with caution. Keywords--Enzyme kinetics, Dicoumarol, DT-diaphorase, NAD(P)H:(quinone-acceptor), Oxidoreductase
INTRODUCTION
may also bind dicumarol7'8; however, for the sake of simplicity, these are not considered in the following discussion. Dicoumarol is a potent inhibitor of the enzyme DTdiaphorase and K i values of 10 nM have been reported9 for the rat liver cytosolic enzyme with dichlorophenolindophenol (DCPIP), a commonly used electron acceptor. Consequently, inhibition of a metabolic activity by dicoumarol is often taken as an indicator of DT-diaphorase mediated metabolism in biological systems such as isolated hepatocytes, cell cultures, and microsomal preparations, and in processes such as detoxification of quinones, 1°-13 bioreductive activation or deactivation of antitumor quinones,14--ls and quinone and other chemical-induced mutagenicity.4'z9-2° The concentration of dicoumarol used in such experiments is usually kept as low as possible to avoid toxicity, effects on enzymes other than DT-diaphorase 2~ and mitochondrial uncoupling. 22 A recent exchange of notes has highlighted the ambiguities which may arise from reliance on dicoumarolmediated inhibition of metabolism or cytotoxicity in cellular systems as evidence for participation of DT-diaphorase. 23'24 The information obtained from such stud-
D T - d i a p h o r a s e or N A D ( P ) H : ( q u i n o n e - a c c e p t o r ) oxidoreductase (E.C. 1.6.99.2) is an obligate two-electron reductase which catalyzes reduction of a wide variety of substrates including quinones, quinone imines, quinone 2,3-epoxides, azo compounds, and some inorganic ions. 1-4 The enzyme is characterized by its ability to utilize either NADH or NAD(P)H as a source of reducing equivalents and by its inhibition by dicoumarol. 5'6 DT-diaphorase functions via a ping pong bibi kinetic mechanism, wherein the reduced pyridine nucleotide binds, reduces the flavin cofactor, and the oxidized pyridine nucleotide is released, prior to binding of the quinone substrate. 7'8 Dicoumarol binds to the oxidized form of the enzyme, competitively versus the reduced pyridine nucleotide7"9 (Scheme 1). In addition, substrate inhibition is observed at high concentrations for certain electron acceptors due to formation of oxidized enzyme--electron acceptor complexes and these
§Address correspondence to David Ross, School of Pharmacy, Campus Box 297, University of Colorado, Boulder, CO 80309. 77
78
P.C. PREUSCHet
ies can only be viewed at best as indirect and further experiments should he designed to test the involvement of DT-diaphorase in a cellular process, preferably by using purified enzyme in a cell-free system. This recent discussion centered on the role of DT-diaphorase in the bioreductive activation of antitumor quinones such as diaziquone (AZQ) and mitomycin C, which has now been clarified. 25'26 One implication of this discussion was that if inhibitory effects were only observed at high concentrations of dicoumarol, then DT-diaphorase was probably not involved and dicoumarol was eliciting its effects through another pathway. Yet, the question of how high a concentration of dicoumarol must be used in such studies to assure adequate inhibition of DT-diaphorase catalyzed metabolism of a particular quinone substrate has not been addressed. MATERIALS AND METHODS
All experimental procedures have been previously described. 25 RESULTS AND DISCUSSION
We recently reported that the antitumor quinone diaziquone (AZQ) is a substrate for purified rat hepatic and human colon carcinoma cell DT-diaphorases 25 as assayed spectrophotometrically and confirmed using HPLC methods. Interestingly, we found that inhibition of AZQ metabolism by purified rat hepatic DT-diaphorase required higher concentrations of dicoumarol than we initially expected. Specifically, we observed only 14% inhibition at 1 IxM, 53% at 2 txM, 77% at 5 t~M, and >99% at 10 ~M dicoumarol. In contrast, metabolism of the well-known substrate DCPIP assayed spectrophotometrically at 600 nm was inhibited >99% at 1 ~M dicoumarol. DCPIP is one of the best known substrates for DT-diaphorase and on the basis of V/K ratios for the purified rat hepatic enzyme, AZQ is 500 times less efficient a substrate. It has also been noted that reduction of vitamin K1, a very poor substrate for DTdiaphorase (185-fold less efficient than DCPIP based on V/K ratios), is also highly resistant (40-fold less sensitive) to dicoumarol. 27'28 Ernster et al. 5'6 originally showed that the concentration of dicoumarol required for half maximal inhibition of rat liver DT-diaphorase can vary when different electron acceptors are used. Thus, there seems to be a difference in the amount of dicoumarol needed for effective inhibition of DT-diaphorase when poor acceptors such as AZQ and vitamin K1 are used versus that needed when good acceptors such as DCPIP and menadione are used. It is not immediately obvious why this should be so, given that dicoumarol inhibits by binding to the reduced pyridine nucleotide binding site of the oxidized form of the en-
al.
zyme, and not to the quinone acceptor site of the reduced form (Scheme 1). The purpose of this note is to highlight the theoretical enzyme kinetic expressions which predict this type of behavior. Initial velocity steady state rate equations describing the kinetic mechanism illustrated in Scheme 1 have been derived by several workers. In the notation of Cleland29: VAB v =
KbA + Ka(1 + I/Ki)B + AB
(l)
where: V = Vmax; A = concentration of the competing substrate (i.e., NADH or NADPH); B = concentration of the uncompetitive substrate (e.g., quinone); Ka, K b = the true Michaelis constants of A and B, respectively; I = concentration of an inhibitor which binds to the oxidized form of the enzyme, only; and K i -- dissociation constant of the oxidized enzyme-inhibitor complex (El). The inhibitor I, is competitive versus the substrate A, and uncompetitive versus the substrate B. In the absence of inhibitor: v =
VAB KbA + KaB +AB
(2)
An expression for the concentration of inhibitor producing 50% inhibition (I5o) may be obtained by setting Equation 1 = 1/2 × Equation 2, and solving for I: 15o
=
Ki
[1 + A/Ka(1
+
Kb/B)]
(3)
Thus, the 15o concentration of inhibitor will be a function of the competing substrate (e.g., NADH) concentration and its Michaelis constant, and less intuitively, of the uncompetitive substrate (e.g., quinone) concentration and its Michaelis constant. The decrease in sensitivity to the inhibitor as the concentration of the competing substrate approaches saturation (A/K a increases) is generally well understood. The effect of concentration and Michaelis constant of the uncompetitive substrate is less well appreciated. For example, if we arbitrarily set A = K a, ( A r K a = 1), then Equation 3 becomes: I5o = 2K i + KiKtfB. The I5o value varies from a minimum of twice the dissociation constant (2Ki) to essentially infinite as K b becomes very large (e.g., poor substrates) or B becomes small. The effect of the uncompetitive substrate term (Kb/B) is multiplied by that of the competing substrate term (A/Ka). Thus, at nearly saturating concentrations of the competing substrate (high A/Ka) as used in some protocols for routine assay of DT-diaphorase (30), the effect of variations in uncompetitive substrate efficiency on inhibitor sensitivity will be magnified. Conversely, at low concentration
Inhibition of DT-diaphorase
79
(NAPH)
(NAD +)
(Quinone)
(Hydroquinone)
A
P
B
Q
ka
kl IRK-1 E --
(EA= EP)
-
F --
[k-3 .
(FB= EQ)
E
El Scheme 1. Ping pong bi-bi kinetic mechanism of action for DT-diaphorase and its inhibition by dicoumarol. The kinetic mechanism for a ping pong bi-bi enzymatic reaction, in the case of DT-diaphorase involving stable oxidized (E) and reduced (F) flavin cofactor forms of the enzyme. (EA = EP) and (FB = EQ) are the transient enzyme-substrate complexes formed on binding of the "first" substrate (A) to enzyme form E, and of the "second" substrate (B) to enzyme form F, respectively. The rate constants shown are for individual reaction steps, Ki is the dissociation constant for the oxidized enzyme-inhibitor complex (El). The inhibitor does not interact with other forms of the enzyme, and therefore, is competitive versus substrate A, uncompetitive versus substrate B. When P = 0, Q = 0, that is, under initial velocity conditions, product release is irreversible, and the rate equation describing this system is given in Equation 1.
of A (or low A/K~), the effect of variations in Kb/B disappears, in fact in this limit only, 15o approaches K i. The identity of the uncompetitive substrate (B) further affects the sensitivity of the enzyme to inhibition by its contribution to the value of the Michaelis constant for A (Ka). Expressed in terms of the rate constants for each step of the mechanism shown in Scheme 1, the experimentally determinable kinetic constants of equation 1 are all combinations of rates which will vary with the identity of both substrates A and B3I:
Ka = (k_ 1 + k2)k4 kl(k 2 + k 4)
Kb = k2(k-3 + k4) (k2 + k4)k 3
V = (k2k4)[Et°tal] (k 2 + k4) The definition of K a includes the term k 4, the rate of turnover of the reduced enzyme-acceptor complex (F-B) to generate the second product (Q) and regenerate the oxidized form of the enzyme. Thus K a is the K M of A with a particular B as substrate, and may differ for various quinone acceptors. For slow turnover acceptors, the K M for N A D H may be significantly lower, than for better substrates, hence for assays at a typical high concentration of NADH, the enzyme may be effectively saturated with NADH. Under these conditions, the effect of the Kb/B term on the sensitivity of the reaction to inhibitor as shown in Equation 3 would be magnified. Another way of considering the effect of the uncompetitive substrate is in terms of the concentrations of the various forms of the enzyme. In the absence of the inhibitor, the fraction of total enzyme in oxidized (E) and reduced (F) forms is given by the following equations29:
KaB E/Et = KbA + KaB + AB
F/Et =
E/F = Ka
KbA KbA + KaB + AB
B
A Kb The effect of the uncompetitive substrate may be viewed in terms of its effect on the concentration of the form of the enzyme to which the inhibitor binds. For a poor second substrate, most of the enzyme is present in the reduced form (F), or in transient complexes (FB = EQ), and thus unavailable as the oxidized enzyme (E) to form complexes with the inhibitor. Hence inhibition of the reduction of poor substrates (high Kb) will require a higher concentration of inhibitor than that for good (low K b) substrates. The value of K i in the above equations, and as determined from a study of the rate versus substrate A (NADH) concentration and inhibitor concentration, is a true dissociation constant for the oxidized--enzyme inhibitor complex. It should not vary with either the concentration or identity of either substrate A (e.g., N A D H or NADPH) or substrate B (e.g., one quinone or another). It should be stressed that the concentration of inhibitor required to achieve 50% inhibition (I5o), or similarly, the extent of inhibition to be expected at any given concentration of inhibitor, will be a function of the concentrations of both the reductant, substrate A (e.g., N A D H or NADPH), and electron acceptor, substrate B (e.g., DCPIP, AZQ, vitamin K1, etc.). In the case of AZQ, no evidence of saturation was obtained over the range from (25 to 200 ~M AZQ) at several N A D H concentrations. The apparent K M for N A D H (a function of AZQ concentration) was (5.6 IxM at 200 p~M AZQ). Thus true values for K a (K M of N A D H
80
P.C. l~t:scH et al.
with AZQ as acceptor) and K b (KM of AZQ with NADH as donor) cannot be obtained. However, it is clear that the K b for AZQ must be considerably in excess of the highest concentration tested (i.e., > 200 IxM), and the K a for NADH must be lower than that normally observed with DCPIP as the substrate (true KM --- 88 ~M). Thus a higher concentration of dicoumarol is required for inhibition of DT-diaphorase mediated metabolism of AZQ than for DCPIP. If inhibition of the reaction is to be used as a probe of the involvement of DT-diaphorase in metabolism of such compounds in cells, then a high concentration of dicoumarol must be tolerated to ensure the desired effect. It should be stressed however, that the high concentration of dicoumarol required may have additional effects on cellular processes other than DTdiaphorase. It is clear that the involvement of DT-diaphorase in metabolism cannot be ruled out by the lack of an inhibitory effect of low doses of dicoumarol, unless it is known that such doses do in fact inhibit reduction of the particular electron acceptor under investigation. In summary, using both theoretical and experimental approaches, we have shown that the concentration of dicoumarol required for effective inhibition of DT-diaphorase depends upon the efficiency of the electron acceptor or second substrate. Acknowledgements CA51210.
-
-
This work was supported by NIH grant number
REFERENCES
1. Ernster, L. DT-diaphorase: a historical review. Chemica Scripta 27A:1-13; 1987. 2. Powis, G.; Lee, S. K.; Santone, K. S.; Melder, D. C.; Hodnett, E. M. Quinoneimines as substrates for quinone reductase (NAD(P)H: (quinone-acceptor) oxidoreductase) and the effect of dicumarol on their cytotoxicity. Biochem. Pharmacol. 36:2473-2479; 1987. 3. Brunmark, A.; Cadenas, E.; Lind, C.; Segura-Aguilar, J.; Emster, L. DT-diaphorase catalyzed two-electron reduction of quinone epoxides. Free Radic. Biol. Med. 3:181-188; 1987. 4. DeFlora, S.; Bennicelli, C.; Camoirano, A.; Serra, D.; Hochstein, P. Influence of DT-diaphorase on the mutagenicity of organic and inorganic compounds. Cardinogenesis 9:611-617; 1988. 5. Emster, L.; Ljunggren, M.; Danielson, L. Purification and some properties of a highly dicumarol sensitive liver diaphorase. Biochem. Biophys. Res. Commun. 2:88--92; 1960. 6. Ernster, L.; Danielson, L.; Ljunggren, M. DT-diaphorase I: purification from the soluble fraction of rat liver cytoplasm and properties. Biochim. Biophys. Acta 58:171-188; 1962. 7. Hosoda, S.; Nakamura, W.; Hayashi, K. Properties and reaction mechanism of DT-diaphorase from rat liver. J. Biol. Chem. 249: 6416--6423; 1974. 8. Hollander, P. M.; Bartfai, T.; Gatt, S. Studies on the reaction mechanism of DT-diaphorase: intermediatry plateau and trough regions in the initial velocity versus substrate concentration curves. Arch. Biochem. Biophys. 169:568-576; 1975. 9. Hollander, P. M.; Ernster, L. Studies on the reaction mechanism of DT-diaphorase. Action of dead-end inhibitors and effects of phospholipids. Arch. Biochem. Biophys. 169:560-567; 1975. 10. Thor, H.; Smith, M. T.; Hartzell, P.; Bellomo, G.; Jewell, S. A.; Orrenius, S. The metabolism of menadione by isolated hepatocytes. J. Biol. Chem. 257:12419-12423; 1982.
11. Morrison, H.; Jemstrom, B.; Nordenskjold, M.; Thor, H.; Orrenius, S. Induction of DNA damage by menadione in primary cultures of rat hepatocytes. Biochem. Pharmacol. 33:1761-1769: 1984. 12. Lind, C.; Hochstein, P.; Emster, L. DT-diaphorase as a quinone reductase. A cellular control device against semiquinone and superoxide radical formation. Arch. Biochem. Biophys. 216:178185; 1982. 13. D'Arcy-Doherty, M.; Cohen, G. M.; Smith, M. T. Mechanism of toxic injury to isolated hepatocytes by 1-naphthol. Biochem. Pharmacol. 33:543-549; 1984. 14. Keyes, S. R.; Francasso, P. M.; Heimbrook, D. C.; Rockwell, S.; Sligar, S. G.; Sartorelli, A. C. Role of NADPH:cytochrome c reductase and DT-diaphorase in the biotransformation of mitomycin C. Cancer Res. 44:5638-5643; 1984. 15. Keyes, S. R.; Rockwell, S.: Sartorelli, A. C. Modification of the metabolism and cytotoxicity of bioreductive alkylating agents by dicoumarol in aerobic and hypoxic murine tumor cells. Cancer Res. 49:3310-3313; 1989. 16. Talcott, R. E.; Rosenblum, M.; Levin, V. A. Possible role of DT-diaphorase in the bioactivation of antitumor quinones. Biochem. Biophys. Res. Commun. 111:346-351; 1983. 17. Begleiter, A.; Robotham, E.; Lacey, G.; Leith, M. K. Increased sensitivity of quinone-resistant cells to mitomycin C. Cancer Letters 45:173-176; 1989. 18. Marshall, R. S.; Paterson, M. C.; Rauth, A. M. Deficient activation by a human cell strain leads to mitomycin resistance under aerobic but not hypoxic conditions. Brit. J. Cancer 59:341346; 1989. 19. Chesis, P. L.; Levin, D. E.; Smith, M. T.; Emster, L.; Ames, B. N. Mutagenicity of quinones: pathways of metabolic activation and deactivation. Proc. Natl. Acad. Sci., U.S.A. 81:1696-1700; 1984. 20. Hochstein, P.; Atallah, A. S.; Emster, L. DT-diaphorase and the toxicity of quinones. Status and perspectives. In: Chow, C. K., ed. Cellular antioxidant defense mechanisms. Vol. 2. Boca Raton, FL: CRC Press; 1989. 21. Murray, R. D. H.; Brown, S. A. Biological action of 4-hydroxycoumarins. In: Murray, R. D. H.; Brown, S. A. Naturally occurring coumarins, occurrence, chemisto,, and biochemistry. Chapter 11. 1982. 22. Martius, C.; Nitz-Litzow, D. Uber den wirkungsmechanismus des dicumarols und verwandter verbindungen. Biochem. Biophys. Acta 12:134-140; 1953. 23. Workman, P.; Walton, M. I.; Powis, G.; Schlager, J. J. DT-diaphorase: questionable role in mitomycin C resistance, but a target for novel bioreductive drugs? Brit. J. Cancer 60:800-802; 1989. 24. Marshal, R.; Rauth, A. M.; Patterson, M. Reply to the letter from Workman, et al. Brit. J. Cancer 60:803; 1989. 25. Siegel, D.; Gibson, N. W.; Preusch, P. C.; Ross, D. Metabolism of diaziquone by NAD(P)H:(quinone acceptor) oxidoreductase (DT-diaphorase): Role in diaziquone-induced DNA damage and cytotoxicity in human colon carcinoma cells. Cancer Res. 50: 7293-7300; 1990. 26. Siegel, D.; Gibson, N. W.; Preusch, P. C.; Ross, D. Metabolism of mitomycin C by DT-diaphorase. Role in mitomycin C induced DNA damage and cytotoxicity in human colon carcinoma cells. Cancer Res. 50: 1990. 27. Fasco, M. J.; Principe, L. M. Vitamin KI hydroquinone formation catalyzed by DT-diaphorase. Biochem. Biophys. Res. Commun. 104:187-192; 1982. 28. Preusch, P. C.; Smalley, D. M. Vitamin K1 2,3-epoxide and quinone reduction: mechanism and inhibition. Free Radic. Res. Commun. 8:401-415; 1990. 29. Cleland, W. W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. II. Inhibition: Nomenclatureand theory. Biochim. Biophys. Acta 67:173-187; 1963. 30. Ernster, L. DT-diaphorase. Methods in Enzymol. 10:309--317; 1967. 31. Cornish-Bowden, A. Chapter 6. Two substrate reactions. In: Cornish-Bowden, A. Fundamentals of enzyme kinetics. London: Butterworths; 1979: 99-129.
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0891-5849/91 $3.00 + .00 Copyright ¢~1991 PergamonPress plc
•
Brief Communication A NOTE ON THE INHIBITION OF DT-DIAPHORASE BY DICOUMAROL PETER C. PREUSCH,* DAVID SIEGEL,t NEIL W . GIBSON,~ a n d DAVID Rosst *Department of Chemistry, University of Akron, Akron, OH 44303. Present address: Division of Research Grants, National Institutes of Health, Bethesda, MD 20892; tMolecular Toxicology and Environmental Health Sciences Program, School of Pharmacy, University of Colorado, Boulder, CO 80309; :~Division of Pharmaceutics, School of Pharmacy and Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA (Received 9 January 1991; Accepted 5 February 1991) A b s t r a c t - - T h e participation of DT-diaphorase or NAD(P)H:(quinone acceptor) oxidoreductase (E.C. 1.6.99.2) in metabolism or in events leading to toxicity is often implied on the basis of the inhibitory effects of dicoumarol. DT-diaphorase functions via a ping pong bi-bi kinetic mechanism involving oxidized and reduced flavin forms of the free enzyme. Dicoumarol, a potent (Ki = 10 riM) inhibitor, binds to the oxidized form of the enzyme, competitively versus reduced pyridine nucleotide. Inhibition is effectively complete at 1 p,M dicoumarol in typical studies using DCPIP, one of the best known substrates for the enzyme, as electron acceptor. The antitumor quinone Diaziquone (AZQ) is a poor substrate for DT-diaphorase relative to DCPIP, but effective inhibition of its reduction requires ten-fold higher concentrations of dicoumarol than for inhibition of DCPIP reduction under otherwise similar conditions. The variable inhibition of DT-diaphorase by dicoumarol dependent on the efficiency of the electron acceptor can be explained on the basis of the complete rate equation describing its ping pong type kinetic mechanism. Thus, the concentration of dicoumarol used to inhibit DT-diaphorase must be chosen carefully and consideration should be given to the efficiency of the electron acceptor. The absence of an inhibitory effect using low doses of dicoumarol cannot rule out a reaction mediated by DT-diaphorase. Although higher doses of dicoumarol may be required to inhibit DT-diaphorase mediated metabolism of less efficient electron acceptors, the use of such doses in cells may also affect biochemical processes other than DT-diaphorase and should be approached with caution. Keywords--Enzyme kinetics, Dicoumarol, DT-diaphorase, NAD(P)H:(quinone-acceptor), Oxidoreductase
INTRODUCTION
may also bind dicumarol7'8; however, for the sake of simplicity, these are not considered in the following discussion. Dicoumarol is a potent inhibitor of the enzyme DTdiaphorase and K i values of 10 nM have been reported9 for the rat liver cytosolic enzyme with dichlorophenolindophenol (DCPIP), a commonly used electron acceptor. Consequently, inhibition of a metabolic activity by dicoumarol is often taken as an indicator of DT-diaphorase mediated metabolism in biological systems such as isolated hepatocytes, cell cultures, and microsomal preparations, and in processes such as detoxification of quinones, 1°-13 bioreductive activation or deactivation of antitumor quinones,14--ls and quinone and other chemical-induced mutagenicity.4'z9-2° The concentration of dicoumarol used in such experiments is usually kept as low as possible to avoid toxicity, effects on enzymes other than DT-diaphorase 2~ and mitochondrial uncoupling. 22 A recent exchange of notes has highlighted the ambiguities which may arise from reliance on dicoumarolmediated inhibition of metabolism or cytotoxicity in cellular systems as evidence for participation of DT-diaphorase. 23'24 The information obtained from such stud-
D T - d i a p h o r a s e or N A D ( P ) H : ( q u i n o n e - a c c e p t o r ) oxidoreductase (E.C. 1.6.99.2) is an obligate two-electron reductase which catalyzes reduction of a wide variety of substrates including quinones, quinone imines, quinone 2,3-epoxides, azo compounds, and some inorganic ions. 1-4 The enzyme is characterized by its ability to utilize either NADH or NAD(P)H as a source of reducing equivalents and by its inhibition by dicoumarol. 5'6 DT-diaphorase functions via a ping pong bibi kinetic mechanism, wherein the reduced pyridine nucleotide binds, reduces the flavin cofactor, and the oxidized pyridine nucleotide is released, prior to binding of the quinone substrate. 7'8 Dicoumarol binds to the oxidized form of the enzyme, competitively versus the reduced pyridine nucleotide7"9 (Scheme 1). In addition, substrate inhibition is observed at high concentrations for certain electron acceptors due to formation of oxidized enzyme--electron acceptor complexes and these
§Address correspondence to David Ross, School of Pharmacy, Campus Box 297, University of Colorado, Boulder, CO 80309. 77
78
P.C. PREUSCHet
ies can only be viewed at best as indirect and further experiments should he designed to test the involvement of DT-diaphorase in a cellular process, preferably by using purified enzyme in a cell-free system. This recent discussion centered on the role of DT-diaphorase in the bioreductive activation of antitumor quinones such as diaziquone (AZQ) and mitomycin C, which has now been clarified. 25'26 One implication of this discussion was that if inhibitory effects were only observed at high concentrations of dicoumarol, then DT-diaphorase was probably not involved and dicoumarol was eliciting its effects through another pathway. Yet, the question of how high a concentration of dicoumarol must be used in such studies to assure adequate inhibition of DT-diaphorase catalyzed metabolism of a particular quinone substrate has not been addressed. MATERIALS AND METHODS
All experimental procedures have been previously described. 25 RESULTS AND DISCUSSION
We recently reported that the antitumor quinone diaziquone (AZQ) is a substrate for purified rat hepatic and human colon carcinoma cell DT-diaphorases 25 as assayed spectrophotometrically and confirmed using HPLC methods. Interestingly, we found that inhibition of AZQ metabolism by purified rat hepatic DT-diaphorase required higher concentrations of dicoumarol than we initially expected. Specifically, we observed only 14% inhibition at 1 IxM, 53% at 2 txM, 77% at 5 t~M, and >99% at 10 ~M dicoumarol. In contrast, metabolism of the well-known substrate DCPIP assayed spectrophotometrically at 600 nm was inhibited >99% at 1 ~M dicoumarol. DCPIP is one of the best known substrates for DT-diaphorase and on the basis of V/K ratios for the purified rat hepatic enzyme, AZQ is 500 times less efficient a substrate. It has also been noted that reduction of vitamin K1, a very poor substrate for DTdiaphorase (185-fold less efficient than DCPIP based on V/K ratios), is also highly resistant (40-fold less sensitive) to dicoumarol. 27'28 Ernster et al. 5'6 originally showed that the concentration of dicoumarol required for half maximal inhibition of rat liver DT-diaphorase can vary when different electron acceptors are used. Thus, there seems to be a difference in the amount of dicoumarol needed for effective inhibition of DT-diaphorase when poor acceptors such as AZQ and vitamin K1 are used versus that needed when good acceptors such as DCPIP and menadione are used. It is not immediately obvious why this should be so, given that dicoumarol inhibits by binding to the reduced pyridine nucleotide binding site of the oxidized form of the en-
al.
zyme, and not to the quinone acceptor site of the reduced form (Scheme 1). The purpose of this note is to highlight the theoretical enzyme kinetic expressions which predict this type of behavior. Initial velocity steady state rate equations describing the kinetic mechanism illustrated in Scheme 1 have been derived by several workers. In the notation of Cleland29: VAB v =
KbA + Ka(1 + I/Ki)B + AB
(l)
where: V = Vmax; A = concentration of the competing substrate (i.e., NADH or NADPH); B = concentration of the uncompetitive substrate (e.g., quinone); Ka, K b = the true Michaelis constants of A and B, respectively; I = concentration of an inhibitor which binds to the oxidized form of the enzyme, only; and K i -- dissociation constant of the oxidized enzyme-inhibitor complex (El). The inhibitor I, is competitive versus the substrate A, and uncompetitive versus the substrate B. In the absence of inhibitor: v =
VAB KbA + KaB +AB
(2)
An expression for the concentration of inhibitor producing 50% inhibition (I5o) may be obtained by setting Equation 1 = 1/2 × Equation 2, and solving for I: 15o
=
Ki
[1 + A/Ka(1
+
Kb/B)]
(3)
Thus, the 15o concentration of inhibitor will be a function of the competing substrate (e.g., NADH) concentration and its Michaelis constant, and less intuitively, of the uncompetitive substrate (e.g., quinone) concentration and its Michaelis constant. The decrease in sensitivity to the inhibitor as the concentration of the competing substrate approaches saturation (A/K a increases) is generally well understood. The effect of concentration and Michaelis constant of the uncompetitive substrate is less well appreciated. For example, if we arbitrarily set A = K a, ( A r K a = 1), then Equation 3 becomes: I5o = 2K i + KiKtfB. The I5o value varies from a minimum of twice the dissociation constant (2Ki) to essentially infinite as K b becomes very large (e.g., poor substrates) or B becomes small. The effect of the uncompetitive substrate term (Kb/B) is multiplied by that of the competing substrate term (A/Ka). Thus, at nearly saturating concentrations of the competing substrate (high A/Ka) as used in some protocols for routine assay of DT-diaphorase (30), the effect of variations in uncompetitive substrate efficiency on inhibitor sensitivity will be magnified. Conversely, at low concentration
Inhibition of DT-diaphorase
79
(NAPH)
(NAD +)
(Quinone)
(Hydroquinone)
A
P
B
Q
ka
kl IRK-1 E --
(EA= EP)
-
F --
[k-3 .
(FB= EQ)
E
El Scheme 1. Ping pong bi-bi kinetic mechanism of action for DT-diaphorase and its inhibition by dicoumarol. The kinetic mechanism for a ping pong bi-bi enzymatic reaction, in the case of DT-diaphorase involving stable oxidized (E) and reduced (F) flavin cofactor forms of the enzyme. (EA = EP) and (FB = EQ) are the transient enzyme-substrate complexes formed on binding of the "first" substrate (A) to enzyme form E, and of the "second" substrate (B) to enzyme form F, respectively. The rate constants shown are for individual reaction steps, Ki is the dissociation constant for the oxidized enzyme-inhibitor complex (El). The inhibitor does not interact with other forms of the enzyme, and therefore, is competitive versus substrate A, uncompetitive versus substrate B. When P = 0, Q = 0, that is, under initial velocity conditions, product release is irreversible, and the rate equation describing this system is given in Equation 1.
of A (or low A/K~), the effect of variations in Kb/B disappears, in fact in this limit only, 15o approaches K i. The identity of the uncompetitive substrate (B) further affects the sensitivity of the enzyme to inhibition by its contribution to the value of the Michaelis constant for A (Ka). Expressed in terms of the rate constants for each step of the mechanism shown in Scheme 1, the experimentally determinable kinetic constants of equation 1 are all combinations of rates which will vary with the identity of both substrates A and B3I:
Ka = (k_ 1 + k2)k4 kl(k 2 + k 4)
Kb = k2(k-3 + k4) (k2 + k4)k 3
V = (k2k4)[Et°tal] (k 2 + k4) The definition of K a includes the term k 4, the rate of turnover of the reduced enzyme-acceptor complex (F-B) to generate the second product (Q) and regenerate the oxidized form of the enzyme. Thus K a is the K M of A with a particular B as substrate, and may differ for various quinone acceptors. For slow turnover acceptors, the K M for N A D H may be significantly lower, than for better substrates, hence for assays at a typical high concentration of NADH, the enzyme may be effectively saturated with NADH. Under these conditions, the effect of the Kb/B term on the sensitivity of the reaction to inhibitor as shown in Equation 3 would be magnified. Another way of considering the effect of the uncompetitive substrate is in terms of the concentrations of the various forms of the enzyme. In the absence of the inhibitor, the fraction of total enzyme in oxidized (E) and reduced (F) forms is given by the following equations29:
KaB E/Et = KbA + KaB + AB
F/Et =
E/F = Ka
KbA KbA + KaB + AB
B
A Kb The effect of the uncompetitive substrate may be viewed in terms of its effect on the concentration of the form of the enzyme to which the inhibitor binds. For a poor second substrate, most of the enzyme is present in the reduced form (F), or in transient complexes (FB = EQ), and thus unavailable as the oxidized enzyme (E) to form complexes with the inhibitor. Hence inhibition of the reduction of poor substrates (high Kb) will require a higher concentration of inhibitor than that for good (low K b) substrates. The value of K i in the above equations, and as determined from a study of the rate versus substrate A (NADH) concentration and inhibitor concentration, is a true dissociation constant for the oxidized--enzyme inhibitor complex. It should not vary with either the concentration or identity of either substrate A (e.g., N A D H or NADPH) or substrate B (e.g., one quinone or another). It should be stressed that the concentration of inhibitor required to achieve 50% inhibition (I5o), or similarly, the extent of inhibition to be expected at any given concentration of inhibitor, will be a function of the concentrations of both the reductant, substrate A (e.g., N A D H or NADPH), and electron acceptor, substrate B (e.g., DCPIP, AZQ, vitamin K1, etc.). In the case of AZQ, no evidence of saturation was obtained over the range from (25 to 200 ~M AZQ) at several N A D H concentrations. The apparent K M for N A D H (a function of AZQ concentration) was (5.6 IxM at 200 p~M AZQ). Thus true values for K a (K M of N A D H
80
P.C. l~t:scH et al.
with AZQ as acceptor) and K b (KM of AZQ with NADH as donor) cannot be obtained. However, it is clear that the K b for AZQ must be considerably in excess of the highest concentration tested (i.e., > 200 IxM), and the K a for NADH must be lower than that normally observed with DCPIP as the substrate (true KM --- 88 ~M). Thus a higher concentration of dicoumarol is required for inhibition of DT-diaphorase mediated metabolism of AZQ than for DCPIP. If inhibition of the reaction is to be used as a probe of the involvement of DT-diaphorase in metabolism of such compounds in cells, then a high concentration of dicoumarol must be tolerated to ensure the desired effect. It should be stressed however, that the high concentration of dicoumarol required may have additional effects on cellular processes other than DTdiaphorase. It is clear that the involvement of DT-diaphorase in metabolism cannot be ruled out by the lack of an inhibitory effect of low doses of dicoumarol, unless it is known that such doses do in fact inhibit reduction of the particular electron acceptor under investigation. In summary, using both theoretical and experimental approaches, we have shown that the concentration of dicoumarol required for effective inhibition of DT-diaphorase depends upon the efficiency of the electron acceptor or second substrate. Acknowledgements CA51210.
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This work was supported by NIH grant number
REFERENCES
1. Ernster, L. DT-diaphorase: a historical review. Chemica Scripta 27A:1-13; 1987. 2. Powis, G.; Lee, S. K.; Santone, K. S.; Melder, D. C.; Hodnett, E. M. Quinoneimines as substrates for quinone reductase (NAD(P)H: (quinone-acceptor) oxidoreductase) and the effect of dicumarol on their cytotoxicity. Biochem. Pharmacol. 36:2473-2479; 1987. 3. Brunmark, A.; Cadenas, E.; Lind, C.; Segura-Aguilar, J.; Emster, L. DT-diaphorase catalyzed two-electron reduction of quinone epoxides. Free Radic. Biol. Med. 3:181-188; 1987. 4. DeFlora, S.; Bennicelli, C.; Camoirano, A.; Serra, D.; Hochstein, P. Influence of DT-diaphorase on the mutagenicity of organic and inorganic compounds. Cardinogenesis 9:611-617; 1988. 5. Emster, L.; Ljunggren, M.; Danielson, L. Purification and some properties of a highly dicumarol sensitive liver diaphorase. Biochem. Biophys. Res. Commun. 2:88--92; 1960. 6. Ernster, L.; Danielson, L.; Ljunggren, M. DT-diaphorase I: purification from the soluble fraction of rat liver cytoplasm and properties. Biochim. Biophys. Acta 58:171-188; 1962. 7. Hosoda, S.; Nakamura, W.; Hayashi, K. Properties and reaction mechanism of DT-diaphorase from rat liver. J. Biol. Chem. 249: 6416--6423; 1974. 8. Hollander, P. M.; Bartfai, T.; Gatt, S. Studies on the reaction mechanism of DT-diaphorase: intermediatry plateau and trough regions in the initial velocity versus substrate concentration curves. Arch. Biochem. Biophys. 169:568-576; 1975. 9. Hollander, P. M.; Ernster, L. Studies on the reaction mechanism of DT-diaphorase. Action of dead-end inhibitors and effects of phospholipids. Arch. Biochem. Biophys. 169:560-567; 1975. 10. Thor, H.; Smith, M. T.; Hartzell, P.; Bellomo, G.; Jewell, S. A.; Orrenius, S. The metabolism of menadione by isolated hepatocytes. J. Biol. Chem. 257:12419-12423; 1982.
11. Morrison, H.; Jemstrom, B.; Nordenskjold, M.; Thor, H.; Orrenius, S. Induction of DNA damage by menadione in primary cultures of rat hepatocytes. Biochem. Pharmacol. 33:1761-1769: 1984. 12. Lind, C.; Hochstein, P.; Emster, L. DT-diaphorase as a quinone reductase. A cellular control device against semiquinone and superoxide radical formation. Arch. Biochem. Biophys. 216:178185; 1982. 13. D'Arcy-Doherty, M.; Cohen, G. M.; Smith, M. T. Mechanism of toxic injury to isolated hepatocytes by 1-naphthol. Biochem. Pharmacol. 33:543-549; 1984. 14. Keyes, S. R.; Francasso, P. M.; Heimbrook, D. C.; Rockwell, S.; Sligar, S. G.; Sartorelli, A. C. Role of NADPH:cytochrome c reductase and DT-diaphorase in the biotransformation of mitomycin C. Cancer Res. 44:5638-5643; 1984. 15. Keyes, S. R.; Rockwell, S.: Sartorelli, A. C. Modification of the metabolism and cytotoxicity of bioreductive alkylating agents by dicoumarol in aerobic and hypoxic murine tumor cells. Cancer Res. 49:3310-3313; 1989. 16. Talcott, R. E.; Rosenblum, M.; Levin, V. A. Possible role of DT-diaphorase in the bioactivation of antitumor quinones. Biochem. Biophys. Res. Commun. 111:346-351; 1983. 17. Begleiter, A.; Robotham, E.; Lacey, G.; Leith, M. K. Increased sensitivity of quinone-resistant cells to mitomycin C. Cancer Letters 45:173-176; 1989. 18. Marshall, R. S.; Paterson, M. C.; Rauth, A. M. Deficient activation by a human cell strain leads to mitomycin resistance under aerobic but not hypoxic conditions. Brit. J. Cancer 59:341346; 1989. 19. Chesis, P. L.; Levin, D. E.; Smith, M. T.; Emster, L.; Ames, B. N. Mutagenicity of quinones: pathways of metabolic activation and deactivation. Proc. Natl. Acad. Sci., U.S.A. 81:1696-1700; 1984. 20. Hochstein, P.; Atallah, A. S.; Emster, L. DT-diaphorase and the toxicity of quinones. Status and perspectives. In: Chow, C. K., ed. Cellular antioxidant defense mechanisms. Vol. 2. Boca Raton, FL: CRC Press; 1989. 21. Murray, R. D. H.; Brown, S. A. Biological action of 4-hydroxycoumarins. In: Murray, R. D. H.; Brown, S. A. Naturally occurring coumarins, occurrence, chemisto,, and biochemistry. Chapter 11. 1982. 22. Martius, C.; Nitz-Litzow, D. Uber den wirkungsmechanismus des dicumarols und verwandter verbindungen. Biochem. Biophys. Acta 12:134-140; 1953. 23. Workman, P.; Walton, M. I.; Powis, G.; Schlager, J. J. DT-diaphorase: questionable role in mitomycin C resistance, but a target for novel bioreductive drugs? Brit. J. Cancer 60:800-802; 1989. 24. Marshal, R.; Rauth, A. M.; Patterson, M. Reply to the letter from Workman, et al. Brit. J. Cancer 60:803; 1989. 25. Siegel, D.; Gibson, N. W.; Preusch, P. C.; Ross, D. Metabolism of diaziquone by NAD(P)H:(quinone acceptor) oxidoreductase (DT-diaphorase): Role in diaziquone-induced DNA damage and cytotoxicity in human colon carcinoma cells. Cancer Res. 50: 7293-7300; 1990. 26. Siegel, D.; Gibson, N. W.; Preusch, P. C.; Ross, D. Metabolism of mitomycin C by DT-diaphorase. Role in mitomycin C induced DNA damage and cytotoxicity in human colon carcinoma cells. Cancer Res. 50: 1990. 27. Fasco, M. J.; Principe, L. M. Vitamin KI hydroquinone formation catalyzed by DT-diaphorase. Biochem. Biophys. Res. Commun. 104:187-192; 1982. 28. Preusch, P. C.; Smalley, D. M. Vitamin K1 2,3-epoxide and quinone reduction: mechanism and inhibition. Free Radic. Res. Commun. 8:401-415; 1990. 29. Cleland, W. W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. II. Inhibition: Nomenclatureand theory. Biochim. Biophys. Acta 67:173-187; 1963. 30. Ernster, L. DT-diaphorase. Methods in Enzymol. 10:309--317; 1967. 31. Cornish-Bowden, A. Chapter 6. Two substrate reactions. In: Cornish-Bowden, A. Fundamentals of enzyme kinetics. London: Butterworths; 1979: 99-129.