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Zinc content, metal chelator inactivation and catalytic inhibition of d -lactate dehydrogenase from the barnacle, Balanus nubilus Darwin
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 191, No. 1, November, pp. 59-64, 1978
Zinc Content, Metal Chelator Inactivation and Catalytic Inhibition of o-Lactate Dehydrogenase from the Barnacle, Balanus nubilus Darwin W. ROSS ELLINGTON,’ Department
of Chemistry,
JAMES
R. COOK, AND GEORGE
Pomona College, Claremont,
Received November
California
L. LONG 91711
17, 1977; revised May 31, 1978
Purified NAD-linked n-lactate dehydrogenase from the depressor muscle of the giant barnacle, B&anus nub&s Darwin, is inactivated when incubated with the metal chelators o-phenanthroline and EDTA. M-Phenanthroline and p-phenanthroline, which lack metal chelating ability, are ineffective in inactivating the enzyme. Inactivated enzyme can be reactivated by the addition of zinc ions to the assay mixture. Atomic absorption spectrophotometric analysis of purified B. nub&s n-lactate dehydrogenase revealed that this enzyme contains stoichiometric amounts of zinc (2 g-atoms per mol of subunit), unlike other lactate dehydrogenases, which lack zinc. Zinc appears to be required for maximal catalytic activity. Aromatic, nitrogen-containing metal chelators and their nonchelating analogs are effective instantaneous inhibitors of B. nubilus n-lactate dehydrogenase. These compounds bind at the coenzyme binding site, as the mode of inhibition is distinctly competitive with respect to NADH. The different effects of metal chelators and their nonchelator analogs suggest that time-dependent inactivation (chelation of the enzyme zinc ions) and instantaneous inhibition (competition with NADH binding) have independent mechanisms.
The presence of zinc (Zn”‘) and inactivation by metal chelators have been clearly demonstrated for yeast and horse liver alcohol dehydrogenase (1, 2). Since the dehydrogenases show a great deal of catalytic and structural homology (3), it is reasonable to consider that metals may be integral parts of other dehydrogenases. A preliminary communication reported that rabbit muscle lactate dehydrogenase was sensitive to metal chelators and contained significant levels of zinc (4). Later work showed, however, that L-lactate dehydrogenases from such diverse sources as rat liver (5), pig heart (6), beef muscle, beef heart, chicken muscle and chicken heart (7) contained only trace levels of zinc. Additionally, these enzymes were not inactivated by incubation with metal chelators. During the course of purification of the NAD-linked n-lactate dehydrogenase (EC 1.1.1.28) from the muscle of the giant barnacle, Balanus nubilus Darwin, we observed inactivation of crude cell-free ex-
tracts by incubation with 1.0 mM EDTA, ophenanthroline, 8-hydroxyquinoline and Lu,cr-dipyridyl (8). In this paper, we report further studies on the effects of these metal chelators and their nonchelating analogs on the purified enzyme. The results presented in this communication indicate that B. nubilus n-lactate dehydrogenase is a zinc metalloenzyme and, as a consequence, appears to be a highly divergent NAD-linked lactate dehydrogenase. MATERIALS
AND
METHODS
Enzyme preparation and enzymatic assay. Purified B. nub&s n-lactate dehydrogenase was prepared as described in the previous paper (9). The resulting enzyme was judged homogeneous by a specific activity of 540 enzyme units per mg protein, electrophoresis on starch and polyacrylamide gels, and behavior in the analytical ultracentrifuge. Enzyme activity and protein content were determined as described in the preceding paper (9). The pyruvate reducing assay (3.0 ml, 25°C) contains 0.05 M potassium phosphate (pH 7.5), 0.14 mM NADH, and 6.7 mM sodium pyruvate. The change in absorbance at 340 nm is measured US.time. A distinction is made throughout this report between time-dependent inactivation and instantaneous inhibition. Operationally, time-dependent inactiua-
’ Present address: Department of Zoology, University of Vermont, Burlington, Vt. 05401. 59
0003-9861/78/1911-0059$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
60
ELLINGTON,
COOK,
tion refers to the loss of enzyme activity observed when enzyme which has been pre-incubated under some set of conditions is then assayed by dilution into the above standard assay. Znstuntuneous inhibition refers to the loss of enzyme activity observed when previously unaltered enzyme is assayed in the above standard assay containing an inhibitor. Preparation of buffer and inhibitor solutions. All stock buffer solutions for kinetic studies were treated with DOWEX chelating resin to remove trace metals according to the method of Willard et al. (10). Inhibitors were checked for purity by thin-layer chromatography using three different solvent systems and by melting point determinations. When judged impure, inhibitors were recrystallized or redistilled to achieve the highest possible purity for kinetic studies. All chelators and chelator analogs were dissolved in 0.05 M potassium phosphate buffer (pH 7.5). The pH of the inhibitor solutions was adjusted when necessary. Zinc analyses. For trace metal analyses, all glassware was washed in concentrated nitric acid followed by treatment with 0.03% dithizone in carbon tetrachloride to remove residual trace metal contaminants. Samples of B. nub&s n-lactate dehydrogenase (540 enzyme units per mg protein) and yeast alcohol dehydrogenase (Sigma Chemical Co., crystallized and lyophilized, 335 units per mg protein) were exhaustively dialyzed against 10 mu Tris-acetate buffer (pH 7.0) containing 1.0 mru dithiothreitol. Zinc was determined by atomic absorption spectrophotometry at 213.9 nm on a Varian Techtron AA6 instrument. A standard curve (0.012-0.250 pg zinc/ml) was constructed using zinc acetate dissolved in buffer resulting from the dialysis. The resulting straight line was generated from seven concentration points and had a correlation coefficient of 0.999. Determinations were made with two dilutions of the pure protein samples.
AND
LONG
phenanthroline and then assayed as described in Table I showed 53% of control activity (untreated with o-phenanthroline) when assayed in the presence of 100 PM ZnCL In the same experiment, the addition of 100 PM CoCL, NiCL, or CuC12 yielded no enzyme activity above that resulting from no metal chloride addition. The inactivation by metal chelators and selective reactivation by zinc ions of B. nubilus n-lactate dehydrogenase strongly suggest that this enzyme requires a metal component. Determinations of zinc content were made by atomic absorption spectrophotometry on purified, fully active enzyme preparations which had been dialyzed exhaustively against buffer containing the sulfhydryl protective reagent, dithiothreitol. Sigma yeast alcohol dehydrogenase, treated under identical conditions, was analyzed as a control, as this enzyme has been recently shown by several methods to contain an average of 1.6 g-atoms zinc/m01 of subunit (11). The results of the atomic absorption analyses indicate that B. nubilus n-lactate dehydrogenase when maintained in the presence of sulthydryl protective TABLE
I
THE EFFECT OF INCUBATION WITH oPHENANTHROLINE AND Two OF ITS NONCHELATING ANALOGS ON THE ACTIVITY OF PURIFIED B. nubilus D-LACTATE DEHYDROGENASE.~ Incubation
medium
RESULTS
Purified B. nubilus n-lactate dehydrogenase is time-dependently inactivated by incubation with o-phenanthroline (Table I). Two nonchelating analogs of o-phenanthroline, m-phenanthroline and p-phenanthroline, are ineffective in inactivating the enzyme under the same conditions of incubation. Overnight dialysis of purified enzyme at 3°C in buffer containing 1 mM EDTA also resulted in a 97% loss of enzyme activity. It was previously reported that the addition of Zn2+ to impure enzyme which has been preincubated with o-phenanthroline restores enzyme activity (8). The amount of restoration is inversely dependent upon the time interval of preincubation with o-phenanthroline. Purified enzyme which had been preincubated in 1 mu o-
50 mu potassium phosphate buffer (pH 7.5), 14 mM 2-mercaptoethanol Above with 1.0 mM l,lO-phenanthroline (ortho) Above with 1.0 mM 1,7-phenanthroline*
Enzyme activity W/d 8.50 0.25 9.30’
(meta) Above with 1.0 mu 4,7-phenanthrolineb
8.80’
(para) ’ A small amount of enzyme (-20 pgm) was diluted in buffer solutions containing or lacking phenanthrolines and incubated on ice for 3 hr before enzymatic assay. Phenanthroline concentration in the resulting assay mixtures was 3.3 PM, well below the levels required to elicit instantaneous inhibition of the enzyme. b Gift from Dr. B. L. Vallee, Biophysics Research Laboratory, Peter Bent Brigham Hospital, Boston, Mass. c Considered to be within experimental error of the control.
ZINC
CONTENT
OF BARNACLE
agent and yeast alcohol dehydrogenase contains 2.12 -I- 0.12 (mean and SD of values at two different protein concentrations) and 1.38 + 0.12 g-atoms zinc/m01 of subunit, respectively. Purified B. nubilus n-lactate dehydrogenase is instantaneously inhibited when ophenanthroline is present in the assay mixture (Table II). The concentration that elicits 50% inhibition was calculated to be approximately 2.0 mM. Purified L-lactate dehydrogenase from the tail muscle of the lobster, Homarus americanus, was also observed to be inhibited by o-phenanthroline, although the degree of inhibition was not as pronounced as in the barnacle enzyme. n-Lactate dehydrogenase from the horseshoe crab, Limulus polyphemus, was not significantly affected by this chelator. No catalytic instantaneous inhibition of purified B. nubilus n-lactate dehydrogenase is observed when EDTA is present in the assay mixture at concentrations as high as 30 mu. Slight inhibition of activity (-11%) is observed at 60 mru EDTA. Kinetic experiments with purified B. nubilus n-lactate dehydrogenase reveal that instantaneous inhibition by o-phenanthroline is competitive with resepct to the coenzyme, NADH (Fig. 1). Instantaneous inhibition by o-phenanthroline is of a “mixed” nature with respect to pyruvate since both K,,, and V,- appear to be altered. To gain an insight into the mechanism underlying
D-LACTATE
61
DEHYDROGENASE
instantaneous inhibition by aromatic metal chelators, the apparent inhibition constants (Ki values) for a variety of aromatic metal chelators and their nonchelating analogs were determined, to see if inhibitory capacity was correlated with metal chelating ability. Apparent inhibition constants (Ki values) for metal chelators and their nonchelating analogs are listed in Table III. In all cases, plots of l/v US. inhibitor concentration yielded results which are consistent with competitive inhibition with respect to the coenzyme. It is apparent from the inhibition constants that the nonchelator analogs are as effective as the chelators in instantaneously inhibiting B. nub&s n-lactate dehydrogenase. These observations cast some doubt as to the role of metal chelation in catalytic (instantaneous) inhibition of this enzyme. In addition, 2,9-dimethyl-o-phenanthroline, which is three orders of magnitude less effective than o-phenanthroline as a metal chelator (12,13) had a greater capacity than o-phenanthroline to inhibit the barnacle enzyme.
TABLE II INSTANTANEOUSINHIBITION OF PURIFIED B.nubilus D-LACTATEDEHYDROGENASEBYOPHENANTHROLINE.~ o-Phenanthroline bM) 0 1.2 4.4 6.0 9.2
Enzyme activity Balanus D-LDH
Limulus D-LDH*
20.0 16.0 7.0 2.0 0
29.0
(ELJ/ml) Homarus L-LDH~ 16.0 I
L S 28.0
10.5
a The chelator was present in the assay mixture at the stated concentration. The assay conditions are described in Materials and Methods. b Horseshoe crab, L. polyphemus, muscle b-lactate dehydrogenase and lobster, H. americanus, tail muscle L-lactate dehydrogenase purified in our laboratory.
25
50
FIG. 1. Double reciprocal plot of NADH saturation kinetics for purified B. nubilus D-lactate dehydrogenase in the presence of o-phenanthroliie. Inhibitor concentrations were as follows: A, 0; B, 0.3 mM; C, 0.6 mru; D, 0.9 mM; E, 1.2 mM; and F, 1.5 mM. Pyruvate concentration was 6.7 mhr. The reciprocal of the initial reaction velocity (l/v) is plotted against the reciprocal of the NADH concentration (l/S).
62
ELLINGTON, TABLE
COOK, AND
III
INSTANTANEOUS INHIBITION OF PURIFIED B. nubilus D-LACTATE DEHYDROGENASE BY AROMATIC METAL CHELATORS AND THEIR NONCHELATING ANALOGS.” Compound
Apparent
K,
(M) Quinoline 8-Hydroxyquinoline 8-Methoxyquinoline 3,4-Benzoquinoline 5-Hydroxyquinoline m-Phenanthroline o-Phenanthroline p-Phenanthroline 7,8-Benzoquinoline 2,9-Dimethyl-o-phenanthroline
1.8 x 9.1 x 8.6 x 3.3 x 3.2 x 2.9 x 2.5 x 2.2 x 1.5 x 4.0 x
lo+ 1o-4 W4 1o-4 1o-4 1om4 1O-4 w4 1o-4 lo-”
n Assays were performed using at least two NADH concentrations (l-6 X 10m5M), 6.7 mM sodium pyruvate, and at least six inhibitor concentrations. Apparent inhibitor constants (K, values) were calculated from graphic plots of l/v us. inhibitor concentration.
DISCUSSION
The time-dependent inactivation of B. dehydrogenase by incubation with various metal chelators suggests that these compounds interact by chelation with a metal component in this enzyme. The absence of inactivation by incubation with nonchelating analogs of o-phenanthroline further supports this assertion. Both yeast and horse liver alcohol dehydrogenases are inactivated by incubation with metal chelators. o-Phenanthroline inactivates yeast alcohol dehydrogenase by removal of zinc (1). In contrast, horse liver alcohol dehydrogenase is reversibly inactivated by o-phenanthroline, by binding with catalytic zinc without metal removal (2). Incubation of horse liver alcohol dehydrogenase with another chelator, sodium diethyldithiocarbamate, results in “irreversible” inactivation by removal of zinc from the protein (2). The inactivation of B. nub&s n-lactate dehydrogenase by metal chelators and selective reactivation by zinc ions strongly suggest that this enzyme requires a metal component. Atomic absorption analysis of purified enzyme confirmed this hypothesis, by demonstrationg 2 g-atoms zinc/m01 of subunit (monomeric M,, 36,000). Demonstration of stoichiometric amounts of zinc in yeast alcohol dehydrogenase, which
nubilus n-lactate
LONG
served as a control, indicates the validity of the analytical technique. The zinc content of B. nubilus n-lactate dehydrogenase is at least one order of magnitude higher than the trace content of other lactate dehydrogenases (4-6). B. nub&s n-lactate dehydrogenase also contains 10 cysteine residues per subunit (9), the highest cysteine content yet reported for any lactate dehydrogenase. It is likely that several of these thiol groups are involved in zinc binding, as dialysis against buffers lacking sulfhydryl protective reagents results in a decline in measured zinc content. A number of workers have observed that as zinc is removed from horse liver alcohol dehydrogenase, the number of titratable sulfhydryl groups declines, indicating the important interaction of cysteine residues and zinc binding (14). Zinc in B. nubilus n-lactate dehydrogenase appears to be necessary for maximal catalytic activity, as enzyme samples dialyzed in buffers without sulfhydryl protective reagents had reduced zinc content and substantially lower specific activities. This interpretation is substantiated by the results presented in Table I, which clearly show that the presence of a sulfhydryl protective agent alone is not adequate in maintaining enzymatic activity. The results reported here, however, do not definitely establish this point. Akeson (15) demonstrated that horse liver alcohol dehydrogenase contains two zinc atoms per subunit and that one is clearly involved in catalysis. It is thought that the other zinc atom has a structural role. The two zinc atoms have differential reactivity to chelators, with the catalytic zinc being more easily removed (2). The two zinc atoms per subunit observed in the present study for the barnacle enzyme suggest that a similar functional role for the metals may be present. Instantaneous inhibition by metal chelators is often used as a criterion for the establishment of an enzyme as a metalloenzyme or not. This can lead to misleading conclusions, as pig heart s-malate dehydrogenase (16), beef liver glutamate dehydrogenase (17), and human liver aldehyde dehydrogenase (13), which have been demonstrated to contain only trace levels of zinc, are instantaneously inhibited by a number of aromatic chelators. Non-chelat-
ZINC
CONTENT
OF BARNACLE
ing analogs of these compounds are equally effective inhibitors (13, 16, 17). Even the zinc metalloenzyme, yeast alcohol dehydrogenase, is instantaneously inhibited by aromatic chelators and their nonchelating analogs (18). The mode of inhibition by these compounds seems to be related to their aromatic structure rather than to their metal chelating ability. It appears that the reported instantaneous inhibition of B. nubilus n-lactate dehydrogenase is also mediated by a mechanism different from that operating during the enzyme’s time-dependent inactivation by o-phenanthroline and 8-hydroxyquinoline. The observed lack of time-dependent inactivation by m- and p-phenanthroline of B. nubilus n-lactate dehydrogenase and the absence of instantaneous inhibition by nonaromatic EDTA strongly support this assertion. An important additional point to be made on the basis of these studies is that just as one should not conclude that a protein is a metalloenzyme on the basis of instantaneous inhibition by a metal chelator alone, one should not eliminate the possibility of metal involvement merely on the basis of similar inhibition by nonchelating analogs. The instantaneous inhibition by aromatic chelators and their nonchelating analogs observed in the present study and with the previously mentioned dehydrogenases appears to be mediated by an interaction with the coenzyme binding site, since the mode of inhibition of these compounds is distinctly competitive with respect to the coenzyme (13,16-18). A number of workers have suggested that binding is directed by hydrophobic interactions of these aromatic compounds with the enzyme (16,18). However, quantitative structure-activity relationships derived in this laboratory correlating inhibitor effectiveness and hydrophobicity by the Hansch approach (19) suggest that for B. nub&s n-lactate dehydrogenase, as well as for potato tuber L-lactate dehydrogenase (20), the interaction of these inhibitors is not primarily hydrophobic (21). A similar situation has been reported for the effect of substituted 4-quinoline-3carboxylates on rabbit muscle L-lactate dehydrogenase activity (22). The presence of stoichiometric amounts of zinc in B. nubilus D-lactate dehydrogen-
D-LACTATE
DEHYDROGENASE
63
ase clearly sets this enzyme apart from other D- and L-lactate dehydrogenases and, in fact, other NAD-linked dehydrogenases with the exception of the alcohol dehydrogenases. We have considered the possibility that the barnacle enzyme is a broadly specific alcohol dehydrogenase with the additional capacity to utilize keto acids. The enzyme does have a high capacity to oxidize and reduce a-hydroxybutyrate and cu-ketobutyrate, respectively (9). Purified B. nubilus n-lactate dehydrogenase has no capacity to utilize ethanol when assayed at a wide range of ethanol concentrations under assay conditions optimal for most alcohol dehydrogenases. Since other lactate dehydrogenases appear to have only trace levels of metals, the presence of stoichiometric amounts of zinc in barnacle lactate dehydrogenase suggests that it may be a highly divergent form. The divergent nature of this enzyme is also suggested by its unusual substrate stereospecificity (23) and subunit organization (9). ACKNOWLEDGMENTS
We thank Dr. V. Hodge (Scripps Institution of Oceanography, La Jolla, California) for assistance with the atomic absorption spectrophotometry. This work was supported by grants from the Research Corporation (C-456) and the Public Health Service (NIH GM22868) to GLL. REFERENCES 1. VALLEE, B. L. (1955) in Advances in Protein Chemistry (Anson, M. L., BaiIy, L., and Edsal, J. T., eds.), Vol. 10, pp. 317-384, Academic Press, New York. 2. DRUM, D. E., HARRISON, J. L., TINC KAI, L., BELHUNE, J. L., AND VALLEE, B. L. (1967) Proc. Nut. Acad. Sci. 57, 1434-1440. 3. ROSSMAN,M. G., LILJAS, A., BRANDEN,C. I., AND BANASZAK,L. J. (1975) in The Enzymes (Boyer, P. D., ed.), Vol. 11, pp. 61-102, Academic Press, New York. 4. VALLEE, B. L., AND WACKER, W. E. C. (1956) J. Amer. Chem. Sot. 78,1771-1772. 5. VESTLING, C. S., HSIEH, W. T., TERAYAMA, H., AND BAPTIST, J. N. (1963) Acta Chem. Stand. 17, Suppl. 1, 23-26. 6. PFLEIDERER,G., JECKEL, D., AND WIELAND, TH. (1958) Biochem. Zeit. 330, 296-302. 7. PESCE, A., MCKAY, R. H., STOLZENBACH,F., KAHN, R. D., AND KAPLAN, N. 0. (1964) J. Biol. Chem. 239, 1753-1761. 8. ELLINGTON, W. R., AND LONG, G. L. (1977) Fed. Proc. 36, 634.
64
ELLINGTON,
COOK,
9. ELLINGTON, W. R., AND LONG, G. L. (1978) Arch. Biochem. Biophys. 186.265-274. 10. WILLARD, J. M., DAVIS, J. J., AND WOOD, H. G. (1969) Biochemistry 8,3137-3144. 11. SYTKOWSKI, A. J. (1977) Arch. Biochem. Biophys. 184,505-517. 12. MARTELL, A. E., AND SILLBN, L. G. (1964) in Stability Constants of Metal-Ion Complexes, Special Publication No. 17, The Chemical Society, London. 13. OPPENHEIMER, H. L., GREEN, R. W., AND MCKAY, R. H. (1967) Arch. Biochem. Biophys. 119, 552459. 14. I(KESON, i\. (1964) B&hem. Biophys. Res. Commun. 17, 211-214. 15. MATHEWSON, P. R., YOST, F. J., AND HARRISON, J. H. (1973) Biochim. Biophys. Acta 321, 413-422.
AND
LONG
16. YIELDING, K. L., AND TOMPKINS, G. M. (1962)
Biochim. Biophys. Acta 62,327-331. 17. SIDHU, R. S., AND BLAIR, A. H. (1975) Biochem. J. 151,443-445. 18. ANDERSON, B. M., REYNOLDS, M. L., AND ANDERSON, C. D. (1966) Biochim. Biophys. Acta 113, 235-243. 19. HANSCH, C. (1971) in Drug besign (Arie’ns, E. J., ed.), Vol. 1, pp. 271-342, Academic Press, New York. 20. ROTHE, G. M. (1976) Z. Pflazenphysiol. 79,
384-391. 21. LONG, G. L., COOK, J. R., AND ELLINGTON, W. R. (1978) Experientia, 34, 567-568. 22. YOSHIMOTO, M., AND HANSCH, C. (1976) J. Med. Chem. 19.71-98. 23. LONG, G. L. (1976) Comp. Biochem. Physiol. 65B, 77-84.
Zinc Content, Metal Chelator Inactivation and Catalytic Inhibition of o-Lactate Dehydrogenase from the Barnacle, Balanus nubilus Darwin W. ROSS ELLINGTON,’ Department
of Chemistry,
JAMES
R. COOK, AND GEORGE
Pomona College, Claremont,
Received November
California
L. LONG 91711
17, 1977; revised May 31, 1978
Purified NAD-linked n-lactate dehydrogenase from the depressor muscle of the giant barnacle, B&anus nub&s Darwin, is inactivated when incubated with the metal chelators o-phenanthroline and EDTA. M-Phenanthroline and p-phenanthroline, which lack metal chelating ability, are ineffective in inactivating the enzyme. Inactivated enzyme can be reactivated by the addition of zinc ions to the assay mixture. Atomic absorption spectrophotometric analysis of purified B. nub&s n-lactate dehydrogenase revealed that this enzyme contains stoichiometric amounts of zinc (2 g-atoms per mol of subunit), unlike other lactate dehydrogenases, which lack zinc. Zinc appears to be required for maximal catalytic activity. Aromatic, nitrogen-containing metal chelators and their nonchelating analogs are effective instantaneous inhibitors of B. nubilus n-lactate dehydrogenase. These compounds bind at the coenzyme binding site, as the mode of inhibition is distinctly competitive with respect to NADH. The different effects of metal chelators and their nonchelator analogs suggest that time-dependent inactivation (chelation of the enzyme zinc ions) and instantaneous inhibition (competition with NADH binding) have independent mechanisms.
The presence of zinc (Zn”‘) and inactivation by metal chelators have been clearly demonstrated for yeast and horse liver alcohol dehydrogenase (1, 2). Since the dehydrogenases show a great deal of catalytic and structural homology (3), it is reasonable to consider that metals may be integral parts of other dehydrogenases. A preliminary communication reported that rabbit muscle lactate dehydrogenase was sensitive to metal chelators and contained significant levels of zinc (4). Later work showed, however, that L-lactate dehydrogenases from such diverse sources as rat liver (5), pig heart (6), beef muscle, beef heart, chicken muscle and chicken heart (7) contained only trace levels of zinc. Additionally, these enzymes were not inactivated by incubation with metal chelators. During the course of purification of the NAD-linked n-lactate dehydrogenase (EC 1.1.1.28) from the muscle of the giant barnacle, Balanus nubilus Darwin, we observed inactivation of crude cell-free ex-
tracts by incubation with 1.0 mM EDTA, ophenanthroline, 8-hydroxyquinoline and Lu,cr-dipyridyl (8). In this paper, we report further studies on the effects of these metal chelators and their nonchelating analogs on the purified enzyme. The results presented in this communication indicate that B. nubilus n-lactate dehydrogenase is a zinc metalloenzyme and, as a consequence, appears to be a highly divergent NAD-linked lactate dehydrogenase. MATERIALS
AND
METHODS
Enzyme preparation and enzymatic assay. Purified B. nub&s n-lactate dehydrogenase was prepared as described in the previous paper (9). The resulting enzyme was judged homogeneous by a specific activity of 540 enzyme units per mg protein, electrophoresis on starch and polyacrylamide gels, and behavior in the analytical ultracentrifuge. Enzyme activity and protein content were determined as described in the preceding paper (9). The pyruvate reducing assay (3.0 ml, 25°C) contains 0.05 M potassium phosphate (pH 7.5), 0.14 mM NADH, and 6.7 mM sodium pyruvate. The change in absorbance at 340 nm is measured US.time. A distinction is made throughout this report between time-dependent inactivation and instantaneous inhibition. Operationally, time-dependent inactiua-
’ Present address: Department of Zoology, University of Vermont, Burlington, Vt. 05401. 59
0003-9861/78/1911-0059$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
60
ELLINGTON,
COOK,
tion refers to the loss of enzyme activity observed when enzyme which has been pre-incubated under some set of conditions is then assayed by dilution into the above standard assay. Znstuntuneous inhibition refers to the loss of enzyme activity observed when previously unaltered enzyme is assayed in the above standard assay containing an inhibitor. Preparation of buffer and inhibitor solutions. All stock buffer solutions for kinetic studies were treated with DOWEX chelating resin to remove trace metals according to the method of Willard et al. (10). Inhibitors were checked for purity by thin-layer chromatography using three different solvent systems and by melting point determinations. When judged impure, inhibitors were recrystallized or redistilled to achieve the highest possible purity for kinetic studies. All chelators and chelator analogs were dissolved in 0.05 M potassium phosphate buffer (pH 7.5). The pH of the inhibitor solutions was adjusted when necessary. Zinc analyses. For trace metal analyses, all glassware was washed in concentrated nitric acid followed by treatment with 0.03% dithizone in carbon tetrachloride to remove residual trace metal contaminants. Samples of B. nub&s n-lactate dehydrogenase (540 enzyme units per mg protein) and yeast alcohol dehydrogenase (Sigma Chemical Co., crystallized and lyophilized, 335 units per mg protein) were exhaustively dialyzed against 10 mu Tris-acetate buffer (pH 7.0) containing 1.0 mru dithiothreitol. Zinc was determined by atomic absorption spectrophotometry at 213.9 nm on a Varian Techtron AA6 instrument. A standard curve (0.012-0.250 pg zinc/ml) was constructed using zinc acetate dissolved in buffer resulting from the dialysis. The resulting straight line was generated from seven concentration points and had a correlation coefficient of 0.999. Determinations were made with two dilutions of the pure protein samples.
AND
LONG
phenanthroline and then assayed as described in Table I showed 53% of control activity (untreated with o-phenanthroline) when assayed in the presence of 100 PM ZnCL In the same experiment, the addition of 100 PM CoCL, NiCL, or CuC12 yielded no enzyme activity above that resulting from no metal chloride addition. The inactivation by metal chelators and selective reactivation by zinc ions of B. nubilus n-lactate dehydrogenase strongly suggest that this enzyme requires a metal component. Determinations of zinc content were made by atomic absorption spectrophotometry on purified, fully active enzyme preparations which had been dialyzed exhaustively against buffer containing the sulfhydryl protective reagent, dithiothreitol. Sigma yeast alcohol dehydrogenase, treated under identical conditions, was analyzed as a control, as this enzyme has been recently shown by several methods to contain an average of 1.6 g-atoms zinc/m01 of subunit (11). The results of the atomic absorption analyses indicate that B. nubilus n-lactate dehydrogenase when maintained in the presence of sulthydryl protective TABLE
I
THE EFFECT OF INCUBATION WITH oPHENANTHROLINE AND Two OF ITS NONCHELATING ANALOGS ON THE ACTIVITY OF PURIFIED B. nubilus D-LACTATE DEHYDROGENASE.~ Incubation
medium
RESULTS
Purified B. nubilus n-lactate dehydrogenase is time-dependently inactivated by incubation with o-phenanthroline (Table I). Two nonchelating analogs of o-phenanthroline, m-phenanthroline and p-phenanthroline, are ineffective in inactivating the enzyme under the same conditions of incubation. Overnight dialysis of purified enzyme at 3°C in buffer containing 1 mM EDTA also resulted in a 97% loss of enzyme activity. It was previously reported that the addition of Zn2+ to impure enzyme which has been preincubated with o-phenanthroline restores enzyme activity (8). The amount of restoration is inversely dependent upon the time interval of preincubation with o-phenanthroline. Purified enzyme which had been preincubated in 1 mu o-
50 mu potassium phosphate buffer (pH 7.5), 14 mM 2-mercaptoethanol Above with 1.0 mM l,lO-phenanthroline (ortho) Above with 1.0 mM 1,7-phenanthroline*
Enzyme activity W/d 8.50 0.25 9.30’
(meta) Above with 1.0 mu 4,7-phenanthrolineb
8.80’
(para) ’ A small amount of enzyme (-20 pgm) was diluted in buffer solutions containing or lacking phenanthrolines and incubated on ice for 3 hr before enzymatic assay. Phenanthroline concentration in the resulting assay mixtures was 3.3 PM, well below the levels required to elicit instantaneous inhibition of the enzyme. b Gift from Dr. B. L. Vallee, Biophysics Research Laboratory, Peter Bent Brigham Hospital, Boston, Mass. c Considered to be within experimental error of the control.
ZINC
CONTENT
OF BARNACLE
agent and yeast alcohol dehydrogenase contains 2.12 -I- 0.12 (mean and SD of values at two different protein concentrations) and 1.38 + 0.12 g-atoms zinc/m01 of subunit, respectively. Purified B. nubilus n-lactate dehydrogenase is instantaneously inhibited when ophenanthroline is present in the assay mixture (Table II). The concentration that elicits 50% inhibition was calculated to be approximately 2.0 mM. Purified L-lactate dehydrogenase from the tail muscle of the lobster, Homarus americanus, was also observed to be inhibited by o-phenanthroline, although the degree of inhibition was not as pronounced as in the barnacle enzyme. n-Lactate dehydrogenase from the horseshoe crab, Limulus polyphemus, was not significantly affected by this chelator. No catalytic instantaneous inhibition of purified B. nubilus n-lactate dehydrogenase is observed when EDTA is present in the assay mixture at concentrations as high as 30 mu. Slight inhibition of activity (-11%) is observed at 60 mru EDTA. Kinetic experiments with purified B. nubilus n-lactate dehydrogenase reveal that instantaneous inhibition by o-phenanthroline is competitive with resepct to the coenzyme, NADH (Fig. 1). Instantaneous inhibition by o-phenanthroline is of a “mixed” nature with respect to pyruvate since both K,,, and V,- appear to be altered. To gain an insight into the mechanism underlying
D-LACTATE
61
DEHYDROGENASE
instantaneous inhibition by aromatic metal chelators, the apparent inhibition constants (Ki values) for a variety of aromatic metal chelators and their nonchelating analogs were determined, to see if inhibitory capacity was correlated with metal chelating ability. Apparent inhibition constants (Ki values) for metal chelators and their nonchelating analogs are listed in Table III. In all cases, plots of l/v US. inhibitor concentration yielded results which are consistent with competitive inhibition with respect to the coenzyme. It is apparent from the inhibition constants that the nonchelator analogs are as effective as the chelators in instantaneously inhibiting B. nub&s n-lactate dehydrogenase. These observations cast some doubt as to the role of metal chelation in catalytic (instantaneous) inhibition of this enzyme. In addition, 2,9-dimethyl-o-phenanthroline, which is three orders of magnitude less effective than o-phenanthroline as a metal chelator (12,13) had a greater capacity than o-phenanthroline to inhibit the barnacle enzyme.
TABLE II INSTANTANEOUSINHIBITION OF PURIFIED B.nubilus D-LACTATEDEHYDROGENASEBYOPHENANTHROLINE.~ o-Phenanthroline bM) 0 1.2 4.4 6.0 9.2
Enzyme activity Balanus D-LDH
Limulus D-LDH*
20.0 16.0 7.0 2.0 0
29.0
(ELJ/ml) Homarus L-LDH~ 16.0 I
L S 28.0
10.5
a The chelator was present in the assay mixture at the stated concentration. The assay conditions are described in Materials and Methods. b Horseshoe crab, L. polyphemus, muscle b-lactate dehydrogenase and lobster, H. americanus, tail muscle L-lactate dehydrogenase purified in our laboratory.
25
50
FIG. 1. Double reciprocal plot of NADH saturation kinetics for purified B. nubilus D-lactate dehydrogenase in the presence of o-phenanthroliie. Inhibitor concentrations were as follows: A, 0; B, 0.3 mM; C, 0.6 mru; D, 0.9 mM; E, 1.2 mM; and F, 1.5 mM. Pyruvate concentration was 6.7 mhr. The reciprocal of the initial reaction velocity (l/v) is plotted against the reciprocal of the NADH concentration (l/S).
62
ELLINGTON, TABLE
COOK, AND
III
INSTANTANEOUS INHIBITION OF PURIFIED B. nubilus D-LACTATE DEHYDROGENASE BY AROMATIC METAL CHELATORS AND THEIR NONCHELATING ANALOGS.” Compound
Apparent
K,
(M) Quinoline 8-Hydroxyquinoline 8-Methoxyquinoline 3,4-Benzoquinoline 5-Hydroxyquinoline m-Phenanthroline o-Phenanthroline p-Phenanthroline 7,8-Benzoquinoline 2,9-Dimethyl-o-phenanthroline
1.8 x 9.1 x 8.6 x 3.3 x 3.2 x 2.9 x 2.5 x 2.2 x 1.5 x 4.0 x
lo+ 1o-4 W4 1o-4 1o-4 1om4 1O-4 w4 1o-4 lo-”
n Assays were performed using at least two NADH concentrations (l-6 X 10m5M), 6.7 mM sodium pyruvate, and at least six inhibitor concentrations. Apparent inhibitor constants (K, values) were calculated from graphic plots of l/v us. inhibitor concentration.
DISCUSSION
The time-dependent inactivation of B. dehydrogenase by incubation with various metal chelators suggests that these compounds interact by chelation with a metal component in this enzyme. The absence of inactivation by incubation with nonchelating analogs of o-phenanthroline further supports this assertion. Both yeast and horse liver alcohol dehydrogenases are inactivated by incubation with metal chelators. o-Phenanthroline inactivates yeast alcohol dehydrogenase by removal of zinc (1). In contrast, horse liver alcohol dehydrogenase is reversibly inactivated by o-phenanthroline, by binding with catalytic zinc without metal removal (2). Incubation of horse liver alcohol dehydrogenase with another chelator, sodium diethyldithiocarbamate, results in “irreversible” inactivation by removal of zinc from the protein (2). The inactivation of B. nub&s n-lactate dehydrogenase by metal chelators and selective reactivation by zinc ions strongly suggest that this enzyme requires a metal component. Atomic absorption analysis of purified enzyme confirmed this hypothesis, by demonstrationg 2 g-atoms zinc/m01 of subunit (monomeric M,, 36,000). Demonstration of stoichiometric amounts of zinc in yeast alcohol dehydrogenase, which
nubilus n-lactate
LONG
served as a control, indicates the validity of the analytical technique. The zinc content of B. nubilus n-lactate dehydrogenase is at least one order of magnitude higher than the trace content of other lactate dehydrogenases (4-6). B. nub&s n-lactate dehydrogenase also contains 10 cysteine residues per subunit (9), the highest cysteine content yet reported for any lactate dehydrogenase. It is likely that several of these thiol groups are involved in zinc binding, as dialysis against buffers lacking sulfhydryl protective reagents results in a decline in measured zinc content. A number of workers have observed that as zinc is removed from horse liver alcohol dehydrogenase, the number of titratable sulfhydryl groups declines, indicating the important interaction of cysteine residues and zinc binding (14). Zinc in B. nubilus n-lactate dehydrogenase appears to be necessary for maximal catalytic activity, as enzyme samples dialyzed in buffers without sulfhydryl protective reagents had reduced zinc content and substantially lower specific activities. This interpretation is substantiated by the results presented in Table I, which clearly show that the presence of a sulfhydryl protective agent alone is not adequate in maintaining enzymatic activity. The results reported here, however, do not definitely establish this point. Akeson (15) demonstrated that horse liver alcohol dehydrogenase contains two zinc atoms per subunit and that one is clearly involved in catalysis. It is thought that the other zinc atom has a structural role. The two zinc atoms have differential reactivity to chelators, with the catalytic zinc being more easily removed (2). The two zinc atoms per subunit observed in the present study for the barnacle enzyme suggest that a similar functional role for the metals may be present. Instantaneous inhibition by metal chelators is often used as a criterion for the establishment of an enzyme as a metalloenzyme or not. This can lead to misleading conclusions, as pig heart s-malate dehydrogenase (16), beef liver glutamate dehydrogenase (17), and human liver aldehyde dehydrogenase (13), which have been demonstrated to contain only trace levels of zinc, are instantaneously inhibited by a number of aromatic chelators. Non-chelat-
ZINC
CONTENT
OF BARNACLE
ing analogs of these compounds are equally effective inhibitors (13, 16, 17). Even the zinc metalloenzyme, yeast alcohol dehydrogenase, is instantaneously inhibited by aromatic chelators and their nonchelating analogs (18). The mode of inhibition by these compounds seems to be related to their aromatic structure rather than to their metal chelating ability. It appears that the reported instantaneous inhibition of B. nubilus n-lactate dehydrogenase is also mediated by a mechanism different from that operating during the enzyme’s time-dependent inactivation by o-phenanthroline and 8-hydroxyquinoline. The observed lack of time-dependent inactivation by m- and p-phenanthroline of B. nubilus n-lactate dehydrogenase and the absence of instantaneous inhibition by nonaromatic EDTA strongly support this assertion. An important additional point to be made on the basis of these studies is that just as one should not conclude that a protein is a metalloenzyme on the basis of instantaneous inhibition by a metal chelator alone, one should not eliminate the possibility of metal involvement merely on the basis of similar inhibition by nonchelating analogs. The instantaneous inhibition by aromatic chelators and their nonchelating analogs observed in the present study and with the previously mentioned dehydrogenases appears to be mediated by an interaction with the coenzyme binding site, since the mode of inhibition of these compounds is distinctly competitive with respect to the coenzyme (13,16-18). A number of workers have suggested that binding is directed by hydrophobic interactions of these aromatic compounds with the enzyme (16,18). However, quantitative structure-activity relationships derived in this laboratory correlating inhibitor effectiveness and hydrophobicity by the Hansch approach (19) suggest that for B. nub&s n-lactate dehydrogenase, as well as for potato tuber L-lactate dehydrogenase (20), the interaction of these inhibitors is not primarily hydrophobic (21). A similar situation has been reported for the effect of substituted 4-quinoline-3carboxylates on rabbit muscle L-lactate dehydrogenase activity (22). The presence of stoichiometric amounts of zinc in B. nubilus D-lactate dehydrogen-
D-LACTATE
DEHYDROGENASE
63
ase clearly sets this enzyme apart from other D- and L-lactate dehydrogenases and, in fact, other NAD-linked dehydrogenases with the exception of the alcohol dehydrogenases. We have considered the possibility that the barnacle enzyme is a broadly specific alcohol dehydrogenase with the additional capacity to utilize keto acids. The enzyme does have a high capacity to oxidize and reduce a-hydroxybutyrate and cu-ketobutyrate, respectively (9). Purified B. nubilus n-lactate dehydrogenase has no capacity to utilize ethanol when assayed at a wide range of ethanol concentrations under assay conditions optimal for most alcohol dehydrogenases. Since other lactate dehydrogenases appear to have only trace levels of metals, the presence of stoichiometric amounts of zinc in barnacle lactate dehydrogenase suggests that it may be a highly divergent form. The divergent nature of this enzyme is also suggested by its unusual substrate stereospecificity (23) and subunit organization (9). ACKNOWLEDGMENTS
We thank Dr. V. Hodge (Scripps Institution of Oceanography, La Jolla, California) for assistance with the atomic absorption spectrophotometry. This work was supported by grants from the Research Corporation (C-456) and the Public Health Service (NIH GM22868) to GLL. REFERENCES 1. VALLEE, B. L. (1955) in Advances in Protein Chemistry (Anson, M. L., BaiIy, L., and Edsal, J. T., eds.), Vol. 10, pp. 317-384, Academic Press, New York. 2. DRUM, D. E., HARRISON, J. L., TINC KAI, L., BELHUNE, J. L., AND VALLEE, B. L. (1967) Proc. Nut. Acad. Sci. 57, 1434-1440. 3. ROSSMAN,M. G., LILJAS, A., BRANDEN,C. I., AND BANASZAK,L. J. (1975) in The Enzymes (Boyer, P. D., ed.), Vol. 11, pp. 61-102, Academic Press, New York. 4. VALLEE, B. L., AND WACKER, W. E. C. (1956) J. Amer. Chem. Sot. 78,1771-1772. 5. VESTLING, C. S., HSIEH, W. T., TERAYAMA, H., AND BAPTIST, J. N. (1963) Acta Chem. Stand. 17, Suppl. 1, 23-26. 6. PFLEIDERER,G., JECKEL, D., AND WIELAND, TH. (1958) Biochem. Zeit. 330, 296-302. 7. PESCE, A., MCKAY, R. H., STOLZENBACH,F., KAHN, R. D., AND KAPLAN, N. 0. (1964) J. Biol. Chem. 239, 1753-1761. 8. ELLINGTON, W. R., AND LONG, G. L. (1977) Fed. Proc. 36, 634.
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9. ELLINGTON, W. R., AND LONG, G. L. (1978) Arch. Biochem. Biophys. 186.265-274. 10. WILLARD, J. M., DAVIS, J. J., AND WOOD, H. G. (1969) Biochemistry 8,3137-3144. 11. SYTKOWSKI, A. J. (1977) Arch. Biochem. Biophys. 184,505-517. 12. MARTELL, A. E., AND SILLBN, L. G. (1964) in Stability Constants of Metal-Ion Complexes, Special Publication No. 17, The Chemical Society, London. 13. OPPENHEIMER, H. L., GREEN, R. W., AND MCKAY, R. H. (1967) Arch. Biochem. Biophys. 119, 552459. 14. I(KESON, i\. (1964) B&hem. Biophys. Res. Commun. 17, 211-214. 15. MATHEWSON, P. R., YOST, F. J., AND HARRISON, J. H. (1973) Biochim. Biophys. Acta 321, 413-422.
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16. YIELDING, K. L., AND TOMPKINS, G. M. (1962)
Biochim. Biophys. Acta 62,327-331. 17. SIDHU, R. S., AND BLAIR, A. H. (1975) Biochem. J. 151,443-445. 18. ANDERSON, B. M., REYNOLDS, M. L., AND ANDERSON, C. D. (1966) Biochim. Biophys. Acta 113, 235-243. 19. HANSCH, C. (1971) in Drug besign (Arie’ns, E. J., ed.), Vol. 1, pp. 271-342, Academic Press, New York. 20. ROTHE, G. M. (1976) Z. Pflazenphysiol. 79,
384-391. 21. LONG, G. L., COOK, J. R., AND ELLINGTON, W. R. (1978) Experientia, 34, 567-568. 22. YOSHIMOTO, M., AND HANSCH, C. (1976) J. Med. Chem. 19.71-98. 23. LONG, G. L. (1976) Comp. Biochem. Physiol. 65B, 77-84.