Catechol 0-methy leansf erase. 1
ings of the 9th Symposium on Peptide Chemistry, N. Yanaihara,Ed., Protein Research Foundation, Osaka, Japan, 1972, p 90. (12) P. Rivaille, A. Robinson, M. Kamen, and G. Milhaud, Helv. Chim. Acta, 54, 296 (1971). (13) A. V. Schally, A. Arimura, W. H. Carter, T. W. Redding, R. Geiger, W. KBnig, H. Wissman, G. Jaeger, J. Sandow, N. Yanaikara, C. Yanaihara, T. Hashimoto, and M. Sakagami, Biochem. Biophys. Res. Commun., 48, 366 (1972). (14) K. Hofmann, A. Rheiner, and W. D. Peckham, J. Amer. Chem. Soc., 75, 6083 (1953). (15) G. W. Anderson, J. E. Zimmerman, and F. M. Callahan, ibid., 86,1839 (1964). (16) K. Hofmann, A. JBhl, A. E. Furlenmeier, and H. Kappler, ibid., 79,1636 (1957). (17) E. D. Nicolaides and H. A. DeWald, J. Org. Chem., 28, 1926 (1963). (18) N. Yanaihara, M. Sekiya, K. Taka@,H. Kato, M. Ichimura, and T. Nagao, Chem. Pharm. Bull., 15,110 (1966). (19) J. Pless and R. A. Boissonnas, Helv. Chim. Acta, 46, 1609 (1963). (20) R. W. Holley and E. Sondheimer,J. Amer. Chem. Soc., 76,
Journal ofMedicinal Chemistry, 1973, Vol. 16, No. 4
377
1326 (1954). (21) K. Hofmann, F. M. Finn, M. Limetti, J. Montibeller, and G. Zanetti, ibid., 88, 3633 (1966). (22) E. C. Jorgensen, G . C. Windridge, W. Patton, and T. C. Lee, J. Med. Chem., 12,733 (1969). (23) N. Yanaihara, T. Hashimoto, C. Yanaihara, and N. Sakura, Chem. Pharm. Bull., 18,417 (1970). (24) W. L. Bencze and K. Schmid,AnaL Chem., 29,1193 (1957). (25) V. D. Ramirez and S . M. McCann, Endocrinology, 73,193 (1963). (26) G. D. Niswender, A. R. Midgley, Jr., S. E. Monroe, and L. E. Reichert, Jr., Proc. SOC.Exp. Biol. Med., 128, 807 (1968). (27) T. W. Redding, A. V. Schally, A. Arhura, and H. Matsuo, Endocrinology, 90, 764 (1972). (28) U. Zor, T. Kaneko, H. P. G. Schneider, S. M. McCann, I. P. Lowe, G. Bloom, B. Borland, and J. B. Field, Proc. Nat. Acad. Sci. U.S., 63, 918 (1969). (29) A. G. Gilman, ibid., 67, 305 (1970). (30) K. Hofmann, H. Yajima, T. -Y. Liu, and N. Yanaihara, J. Amer. Chem. Soc., 84,4475 (1962). (31) K. Hofmann, T. -Y. Liu, H. Yajima, N. Yanaihara, and S. Lande, ibid., 83, 2294 (1961).
Catechol 0-Methyltransferase. 1 . Kinetics of Tropolone Inhibition Ronald T. Borchardt Department of Biochemistry, McCollum Laboratories, University of Kansas, Lawrence, Kansas 66044. Received October 27, 1972
In an attempt to clarify the mechanism by which tropolones inhibit catechol 0-methyltransferase (COMT), kinetic studies have been performed on COMT purified from rat liver. Rate studies indicate that tropolone is a linear noncompetitive inhibitor with respect to 3,4-dihydroxybenzoic acid (Kii = 247 pM, Kis= 22 p M ) and an uncompetitive inhibitor with respect to S-adenosyl-L-methionine; a complex relationship exists with respect t o magnesium. Similar results were observed with &norepinephrine and 3,4dihydroxyacetophenone as substrates, as well as with 4-methyltropolone and 0-thujaplicin as inhibitors. The patterns of inhibition and the kinetic parameters for tropolone inhibition remained constant through various stages of enzyme purification. The effect of pH on the substrate kinetic parameters (Kmand Vmm) and tropolone inhibition parameters (Kis and Kii) was also investigated. The ability of tropolone to inhibit COMT was found t o decrease with increasing pH which can be attributed to an increase in Kis. This probably reflects the ionization of tropolone rather than dissociation of a group on the enzyme. The Kii for tropolone was observed t o decrease with increasing pH. The significance of this data relative to the mechanism of 0-methylation by COMT is discussed. The inactivation of catecholamines and the detoxification of many zenobiotic catechols is dependent upon the enzyme catechol 0-methyltransferase (COMT) (E.C. 2.1.1.6).+ COMT is a soluble, magnesiumrequiring enzyme which transfers a methyl group from S-adenosylmethionine (SAM) to a catechol substrate resulting in the formation of the meta and para 0-methylated derivatives.I2' Because of the importance of COMT in the extraneuronal inactivation of norepinephrine, inhibition of this route of metabolism has generated considerable research interest. 3-10 Several classes of synthetic inhibitors of COMT have been identified: (a) substrates such as catechol^,^ desmethylpapa~ e r i n eo-dihydroxyphenylacetamide~,~ ,~ and pyrogallol;6 (b) product type inhibitors such as 3-hydroxy-4-methoxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, 3,5-dihydroxy4-me thoxybenzoic acid,' and S-adenosylhomocysteine;8 and (c) dead-end inhibitors such as tropolones' and pyridoxal 5'-Po4.l o Since dead-end inhibitors have proven useful in the elucidation of certain enzyme mechanisms," and since the kinetic mechanism of COMT appears t o be the subject of consider+Abbreviations used are: SAM, S-adenosyl-L-methionine; DHB,
3,4-dihydroxybenzoic acid; I-NE, I-norepinephrine; DHA, 3,4-di-
hydroxyacetophenone; COMT, catechol 0-methyltransferase (E.C. 2.1.1.6); Kjs, Inhibition constant for t h e slope;XC, inhibition constant for the intercept.
able c o n t r o ~ e r s y , it ~ ~was - ~felt ~ ~that ~ an extensive investigation of the kinetics of tropolone inhibition on COMT could provide information relative t o the mechanism of methyl transfer. The present paper reports the results of such an investigation and provides information about the kinetic mechanism for COMT. Results and Discussion
In Vitro Inhibition Patterns. The inhibition patterns toward COMT for three tropolone derivatives were determined using plots of reciprocal velocities against reciprocals of the substrate concentrations. As shown in Figure 1, a noncompetitive type of inhibition was observed when DHB was the variable substrate and tropolone was the inhibitor. Furthermore, this inhibition pattern can be classified as linear noncompetitve since replots of the slopes and intercepts vs. inhibitor concentrations are linear (Figure 2). l5 This type of linear relationship with a dead-end inhibitor provides evidence against the possibility that two or more molecules of the inhibitor add simultaneously to the same form of the enzyme." This, however, does not rule out the possibility that tropolone may be combining with two different forms of the enzyme producing the noncompetitive $c. Creveling, personal communication.
378
Journal of Medicinal Chemistry, 1973, Vol. 16, No. 4
Borchardt Table 1. Inhibition Constants for Various Substituted Tropolones toward COMTQ Inhibition constants. uMb Inhibitor Kii k S.E.M. Kis + S.E.M. Tropolone 2 4 1 + 16 2 2 t 1.0 4-Methyltropolone 143 k 21 8.9 r 1 7 P-Thujapticin 283 ?: 44 10.1 c 2.2 QCOMTwas purified through the calcium phosphate gel step (ref 7) resulting in a preparation which contained 3.53 mg of protein per milliliter with a specific activity of 60.47 nmol/mg of N Kjeldahl/ min (DHB as substrate). Assay conditions: DHB concentrations 40400 SAM concentration 1.O mM; TES buffer pH 7.60 and inhibitor concentrations ranging from 16 to 2 0 0 &f. bData fitted to eq 3.
a;
5
I O
15
2 c
x
~,:DHB:
2 5
I O ' N
Figure 1. Reciprocal plots with DHB as the variable substrate and tropolone as the inhibitor. Assay conditions are the same as in Table I. Vel = nmol of productlmg of N Kjeldahl/min.
I
6 O t
identical with results obtained under saturating conditions (noncompetitive). Further evidence in support of this noncompetitive inhibition comes from data on a series of 8hydroxyquinoline derivatives which have been shown in vitro to be potent noncompetitive inhibitors of COMT. They appear to act at a similar site and by a similar mechanism to that of the tropo1ones.l6 During extensive purification of COMT, the inhibitory constants for tropolone were determined and as shown in Table 111 did not vary significantly. These data as well as data from the meta and para 0-methylation studies of Creveling, et al., and gel electrophoresis studies of this purified enzyme in our laboratory would provide strong evidence that only one enzyme is involved. 5 When SAM was the variable substrate and tropolone the inhibitor, an uncompetitive pattern of inhibition was observed (Figure 3). Identical uncompetitive patterns were observed when 1-NE or DHA were used as substrates, as well as if 4-methyltropolone or P-thujaplicin were used as inhibitors. Utilization of half-maximal concentrations of catechol substrates or Mgz+ rather than saturation conditions of these substrates did not change the inhibition pattern. This uncompetitive pattern of inhibition with varying SAM was found to be independent of pH. This type of uncompetitive dead-end inhibition is characteristically produced by combination of the inhibitor with an enzyme form that occurs after combination of the variable substrate and before release of all the products.lla>bFrom a mechanistic standpoint the uncompetitive pattern observed here would indicate that tropolone was adding to a form of the enzyme after the addition of SAM. A complex relationship exists between Mgz+ concentration, inhibitor concentration, and enzymatic activity. The buffer used in these studies was TES which has been shown to have no magnesium-complexing activity." As shown in Figure 4 the double reciprocal plot of enzyme activity vs. Mgz+at various concentrations of tropolone showed essentially parallel curves. In the absence of inhibitor at very low concentrations of Mg2+little effect on enzyme activity was noted. However, an increased Mg2+concentration produced an activation of reaction rate followed by a progressive inhibition at concentrations of Mgz+ greater than 1 mM. These results are consistent with similar observations by earlier investigator^.^^^ A paralleling effect was observed in the presence of increasing concentrations of tropolone. Even though the interrelationship between Mgz+ and inhibitor is not clear, the presence of inhibition at Mgz+ concentrations greater than that of tropolone and the apparent inability to reverse the inhibition with excess magnesium argues against chelation of 17518
'
O
0
I
Y 0'05
0 10
0 15
0 20
[TROPOLONE], "U
Figure 2. Replot of intercepts and slopes from Figure 1 against tropolone concentration. Equation 1 was used to calculate interce ts and slopes. Where significant +S.E.M. are also shown. intercept; slope.
4,
Q,
pattern. The noncompetitive pattern observed could in fact result from tropolone binding with one form of the enzyme producing competitive kinetics and one form producing uncompetitive kinetics. Using eq 3 for linear noncompetitive kinetics, aKis of 2 2 (k1.0) pM and aKii of 247 (+16) pM for tropolone were calculated. (In linear inhibitions, Kis and Kii represent the concentration of the inhibitor that doubles the slope or intercept term, respectively.) As shown in Table I similar results were observed in the cases of 4-methyltropolone and 0-thujaplicin supporting the general noncompetitive pattern for this class of inhibitors. These results contradict earlier work by Belleau and Burbaqb and Mavrides, et a1.,% in which their preliminary data supported a competitive mechanism for tropolone inhibition. Earlier, however, Belleau and Burba- had proposed a noncompetitive mechanism for this inhibition. T o provide further evidence for a noncompetitive inhibitory mechanism, several other substrates of COMT were studied and the results of tropolone inhibition of their 0-methylation are summarized in Table 11. In each case noncompetitive inhibition was observed. Particularly interesting is the case of I-NE where Kii and Kis for tropolone were of similar magnitude. To eliminate the possible effect of saturation on the enzyme with the nonvaried substrates (SAM and Mg2+) the inhibitory patterns varying DHB at half-maximal concentrations of SAM or Mgz+ were determined and found to be
BR. T. Borchardt, unpublished data.
Journal ofMedicina1 Chemistry, 1973, Vol. 16, No. 4
Catechol 0 - M ethy1transferase. I
319
Table 11. Tropolone Inhibition of COMT" Substrate constantsb Vmax i S.E.M.e 20.7 i 0.28 393 * 43 1.67 f 0.34 8 . 0 4 i 0.37 10.0 t 0.88
Inhibition constants, pMc
K , i s.E.M.~ 90.0 i 4.0
Substrate 3,4-Dihydroxybenzoic acid I-Norepinephrind 3,4-Dihydr oxyacetophenone
Kii i S.E.M.
Ki, i S.E.M.
247 f 16 30.5 i 4.4 270 i 20
22i 1 44.3 i 1.4 50.6 i 4.4
a,
DHA concentrations 16-80 aKinetic assay conditions are the same as those outlined in Table I except 1-NE concentrations of 160-1600 epmol of product /mg of N Kjeldahl/min. fBitartrate salt.
a. and tropolone concentrations 40-200 a. bData fitted to eq 1. CData fitted to eq 3. dpM.
Table 111. Effect of Enzyme Purity on Tropolone Inhibition Inhibition constants, pMbsc S.E.M. Ki, ? S.E.M.
Purification," -fold
Enzyme fraction
Kii i
8 245 f 25 19.1 f 3.2 100,OOOg supernatant 21.4 2.3 30-50% (NH4),S0, 92 237 14 192 271 f 22 18.9 2.7 Sephadex G-25 Calcium phosphate 280 247 16 22.0 * 1.1 ne1 QCOMTwas purified from rat liver as described earlier (ref 7). bAssay conditions (the assay procedures were as described in the Experimental Section): 3,4-dihydroxybenzoic acid was utilized as substrate in concentrations ranging from 40 to 400 SAM concentration, 1.0 M;TES buffer pH 7.60; tropolone concentrations ranging from 40 to 200 pM. CData fitted to eq 3.
* *
* *
a;
0
IS 0
IO 0
5 0 It
[UG*']
20 0
l0"U
X
Figure 4. Reciprocal plots with Mg2+as the variable component and tropolone as the inhibitor. Assay conditions are the same as in Table I except SAM concentration, 1.0 mM. DHB concentration, 2 M.Vel = nmol of product /mg of N Kjeldahl/min.
I)/ 100
I 10
20
3 0
5 0
40
B
01
60 I/[SAM]
X
70
80
90
IO-+M PH
Figure 3. Reciprocal plots with SAM as the variable substrate and tropolone as the inhibitor, Assay conditions are the same as in Table I except SAM concentrations, 24-210 DHB concentration, 2 M.Vel = nmol of productlmg of N Kjeldahl/min.
Figure 5 . Reaction rate of COMT as a function of pH. Assay conditions are the same as in Table I except SAM concentration, 1.0 mM. DHB concentration, 2.0 mM. Mg'+ concentration, 1.2 mM. TES buffer concentration, 40 mM.
the free metal as a mechanism of tropolone inhibition. This does not, however, rule out the possibility that tropolone is bound by an enzyme-Mg" complex. Effect of pH on Kinetic Constants. Considerable information about the functional groups present at or near the absorption pocket on an enzyme can be obtained by studying the variation of the kinetic parameters with pH. According to Dixon," changes in the slope of plots of pKm VS. pH reflect dissociation of ionizing groups that occur in the substrate, the free enzyme, or the enzyme-substrate complex. If the curvature of the pH profile for a particular substrate results from dissociation of groups present at or near the binding site on the enzyme, then other substrates or inhibitors which bind to the same site should show similar inflection points. Since at least part of the inhibition produced by tropolone on COMT can be classified as competitive with respect to the catechol substrate (indicating that tropolone is competing for the same form of the enzyme as the substrate), the effect of pH on the kinetic constants for substrate and
tropolone should provide information relative to similarities or differences in their specific binding sites. The effect of pH on the rate of 0-methylation of DHB by purified COMT is shown in Figure 5. A pH optimum of 7.6 was observed. Previous studies using other substrates have shown a similar pH optimum,12y22 as well as a second optimum at pH 9.7. The first optimum is attributable to the intrinsic properties of the enzyme, whereas the second probably results from an increased concentration of the anionic species of the catechol substrate. Since our interests were strictly in the groups associated with the binding sites on the enzyme, the pH range for our studies was restricted to pH 6.5-9.0. Figure 6 shows the variation of the pKm of DHB with pH. A single inflection point in this pH range was observed indicating dissociation of a group on the enzyme with a pKa = 7.4-7.6. Figure 7 shows the variation of log V,, with pH showing an optimum V, at pH 7.5 with a steady decrease in V,, with increasing pH.
a.
380
Borchardt
Journal of Medicinal Chemistry, 19 73, Vol. 16, No. 4
Table IV. Effect of pH on Inhibition Constants for Tropolonea
s,, 3 50 6 0
80
7 0
90
Pn
Figure 6. pK, for DHB as a function of pH. pK, determined as previously outlined. Data fitted to eq 1 with resulting S.E.M. shown.
l i
5 1
6 00
7 00
8 00
9 00
PH
Figure 7. Log V,,, for COMT as a function of pH. Data fitted to eq 1 with resulting S.E.M. shown.
PHb
InhibitionC
6.83 7.05 I .24 7.42 7.57 7.82 8.04 8.30 8.63
C C C NC NC NC NC NC NC
Inhibition constants, p M __-______ Kii t S.E.M. Kis t S.E.M.
304 t 31 247 f 16 211 i 48 180 t 30 186 t 56 121 235
19.0 i 4.0 19.2 t 2.1 20.3 t 3.7 25.1 f 5.6 22.0 t 1.7 33.2 i 7.1 37.6 f 5.3 39.2 i 4.3 40.0 t 6.7
aKinetic conditions are the same as those outlined in Table I . bTES buffer. CDetermined by inspection of reciprocal plots and if Kii was statistically significant then data fitted to eq 3. C, competitive inhibition and data fitted to eq 2. NC, noncompetitive inhibition and data fitted to eq 3.
competitive kinetics with respect to DHB, whereas at higher pH the kinetic pattern becomes noncompetitive. The decrease in inhibitory ability with increasing pH (Figure 8) results from an increase in Kis for tropolone as shown in Table IV. The increase in Kis, we believe, is attributable to ionization of the inhibitor rather than dissociation of a functional group on the enzyme. Evidence to support this comes from data obtained on a series of 8-hydroxyquinolines which inhibit COMT by the same mechanism and bind to the same sites as tropolone.’6 8-Hydroxyquinoline, which does not itself have a dissociation constant in this pH range, does not show a change in KiS with pH.16 The decrease in the inhibitory ability and the increase in Ki, with increasing pH may reflect the fact that the protonated tropolone species binds better to the enzyme than the anionic species. The decrease in Kii with increasing pH, however, may partly reflect a dissociation of a group on the enzyme, since a similar decrease was observed with 8-hydro~yquinoline.’~ Conclusions
PH
Figure 8. Per cent tropolone inhibition as a function of pH. Assay conditions are the same as in Figure 5 except tropolone concentration, 400 fl.
The effect of pH on tropolone inhibition of COMT is shown in Figure 8. Increases in pH produce a decrease in tropolone’s ability to inhibit 0-methylation of DHB. The observed inflection point at about pH 7.3 could indicate the ionization of a group at the binding site for tropolone; however, the situation is complicated by the fact that ionization of tropolonez3 (pKa = 6.92) also takes place in this region. In an attempt to clarify this situation, the effect of pH on the inhibitory constants for tropolone was determined. The Kii and Ki, are summarized in Table IV. One of the more interesting observations is that at low pH (
