Mechanism-based Inactivation of Dopamine ,&Monooxygenase by ...

THEJOURNAL OF

BIOI.OGICAL CHEMISTRY

8 1984 by The American Society of Biological Chemists, Inc.

Vol. 259, No. 12, Issue of June 25. pp. 7772-7779.1984 Printed in U.S A .

Mechanism-based Inactivation of Dopamine ,&Monooxygenase by ,&Chlorophenethylamine* (Received for publication, December 19,1983)

James B. MangoldS and JudithPollock Klinmang From the Department of Chemistry, University of California, Berkeley, California94720

Functionalization of the&carbon of phenethyl- neurotransmitterbiosynthesis (1-4). Despite considerable amineshas beenshowntoproduce a newclass of scrutiny, a variety of fundamental questions regarding the substratelinhibitor of dopamine 6-monooxygenase. mechanism of oxygen and substrate activation, the role of Whereas both &hydroxy- and 8-chlorophenethylamine copper, andthenature of the enzymeactive siteremain are converted to a-aminoacetophenone at comparable unanswered. rates, only the latter conversion is accompanied by The reaction catalyzed by dopamine 0-monooxygenase inconcomitant enzyme inactivation (Klinman,J. P.,and volves replacement of a hydrogen by a hydroxyl group at the Krueger, M. (1982) Biochemistry 21, 67-75). In the benzylic carbon of dopamine toyield (R)-norepinephrine: present study, the nature of the reactive intermediates leading to dopamine 8-monooxygenase inactivation by ,B-chlorophenethylamine has been investigated employHO ing kinetic deuterium isotope effects and oxygen-18 labeling as tools. Mechanistically significant findings presented herein include: 1) an analysis of primary deuterium isotope effects on turnover, indicating maHO jor differences in rate-determining steps for @-chloroand p-hydroxyphenethylamine hydroxylation, Dkcar= 6.1 and 1.0, respectively; 2) evidence that dehydration of the gem-diol derived from oxygen-18-labeled &hydroxyphenethylamine hydroxylation occurs in a random manner,attributedto dissociation of enzymebound gem-diol prior to a-aminoacetophenone forma-As illustrated, the reaction requires dioxygen and an exogewhich functions toreduce enzyme-bound tion; 3) the observation of a deuterium isotope effect nous electron donor, for @-chlorophenethylamine inactivation,Dkinart = 3.7, Cu(I1) to Cu(1) (5-7). Ascorbic acid has beenidentified in implicating C-H bond cleavage in the inactivation chromaffin vesicles at millimolar concentrations (8) and is process; and 4) the demonstration that a-aminoaceto- believed to serve as the physiologic electron donor via two as exogenous reduc- one-electron steps to generate semidehydroascorbate(9, 10). phenone can replace ascorbic acid P-monooxygenase requirestwo tant in the hydroxylation of tyramine. As discussed, Recent findings that dopamine these findings support the intermediacy of enzyme- copper atomspersubunit formaximal enzymaticactivity bound a-aminoacetophenone in fl-chlorophenethylam- indicate that both reducing equivalents required by the reacus to propose an intramolec- tion stoichiometry can be stored within a single subunit (11, ine inactivation, and lead ular redox reaction to generatea ketone-derived rad- 31). The kinetic order of the interaction of dopamine and ical cation as the dopamine p-monooxygenase-inacti- oxygen with reduced enzyme has been found to be dependent vating species. on both pH and anion activator, varying from a preferred order mechanism (oxygen off first, pH 4.5, uersus dopamine off first, pH 6.6) to a random mechanism at intermediate pH values (12, 13). The enzyme exhibits a broad substrate specificity, which Dopamine P-monooxygenase is a copper-containing monooxygenase which catalyzes the conversion of dopamine to has facilitated mechanistic investigations by the use of subnorepinephrine. Located primarily in chromaffin vesicles of strate analogs (14-16). A number of phenethylamines can stereospecificity for theadrenal medulla andsynaptic vesicles of sympathetic function as substrates and to date the is preservedinallcases (17-19). nerve terminals, thisenzyme has received considerable atten- pro-Rhydrogenremoval tion in recent yearsbecause of its importantrole in hormone/ Recently,May et al. (20) andKlinmanand Krueger (21) reported that pro-S-substituted phenethylamines are hydroxylated by dopamine p-monooxygenase. In these reports, ( S ) octopamine and (S)-P-hydroxyphenethylamine,respectively, * This work was supported by Grant GM 25765 from the National were converted to the corresponding p-keto compounds. Of Institutes of Health. The costs of publication of this article were particular note was the finding that substitutionof the pro-S defrayed in part by the payment of page charges. This article must hydrogen with the leaving group, -C1, produced a new class therefore be hereby marked “aduertisement” in accordance with 18 of mechanism-based inhibitor for dopamine P-monooxygenU.S.C. Section 1734 solely to indicate this fact. ase (21). Present address, Section of Medicinal Chemistry and PharmaInitially, attention was focused on the enzymatic product cognosy, School of Pharmacy, University of Connecticut, Storrs, CT a-aminoacetophenone, 1,as the p-chlorophenethylamine-de06268. § To whom correspondence should be addressed. rived inactivating species:

Inactivation of Dopamine P-Monooxygenme

by P-Chlorophenethylamine

7773

1 ." Synthesis of (R,S)-2-Phenyl-2-hydroxy-[2-2Hjethylamine. HC1 (0However,a-aminoacetophenonedoesnotinactivatedopamethod involved the mine @-monooxygenase under turnover conditions. In addi- fH]Hydroxyphenethylamine .HCl)-This mixed hydride or mixed deuteride reduction of a-aminoacetophenone and @-chlorophenethylaminesboth to yield @-['HI-or p-[2H]hydr~xyphenethylamine,respectively. Tyption, although @-hydroxyyield a-aminoacetophenone as their final product, only the ically, the mixed hydride (deuteride) reagent was prepared by the latter is an inactivator of dopamine @-monooxygenase. Prein- addition of aluminum trichloride (3.675 g, 27.8 mmol) in 25 mlof cubation of enzyme with a-aminoacetophenone in the absence ether to a stirred slurry of lithium aluminum hydride (deuteride) (0.9 g, 27.8 mmol) in 75 ml of ether under N2 atmosphere. After 5 min, of ascorbic acid as reductant produced rapid inactivation, the mixture was cooled to 0 "C and a-aminoacetophenoneHCI (3 g, suggesting that dopamine @-monooxygenase was susceptible 17.5 mmol) wasslowly added in 0.2-g portions. The reaction was to a-aminoacetophenone inhibition only while in the oxidized stirred for 2 h at room temperature and terminated by the sequential Cu(I1) form. T h u s , a workinghypothesis has invoked the addition of water (0.9 ml), 1 N NaOH (0.9 ml), and water (2.7 ml). generation of enzyme-bound ketone, 2 , After filtration, the ether layer was removed. The aqueous layer was adjusted to pH 9 (Na,CO,), saturated with NaCI, and extracted with 0 ether (8 X 50 ml). All ether extracts were pooled and acidified with aqueous HCl, and thesolvent was removedin uacuo. Recrystallization E.Cu(I1). -!--CH.NH: of the residue from absolute ethanol-ether provided the desired 8hydroxyphenethylamine. HC1, m.p. 209-21 1 "C. Yields weregenerally 2 30-50% and the deuterium content at the@-carbonwas greater than as a crucial step toward inactivation. 95%, with no detectable protons by NMR. The present investigation has employed deuterium isotope Synthesis of (R,S)-2-Phenyl-2-~hloro-[2-~H]ethylamine. HC1 (0effects and isotopic labeling as tools to investigate furtherthe fH/Chlorophenethylamine. HCl)-P-['H]- or P-[2H]chlorophenethinactivation mechanism. The results reported herein indicate ylamine.HC1 was prepared by treatment of the appropriate P-hymajor differences in the kinetic mechanisms of @-hydroxyand droxyphenethylamine. HCl precursor with thionyl chloride as described by Barnett et al. (23). The P-['Hlhydroxy compound was /I-chlorophenethylamine, and provide support for t h e inter- obtained both commercially and by mixed hydride reaction of amediacy of 2 in @-chlorophenethylamine inactivation.A key aminoacetophenone as described previously. Product was obtained in replace ascorbic acid 80-95% yield after recrystallization from absolute ethanol-ether, m.p. finding is that a-aminoacetophenone can as electron donor. This important observation leads us t o 164-165 "C. The deuterated product was greater than 95% enriched propose an intramolecular redox reactionof 2 to generateE . with deuterium at the P-carbon, with no detectable protons at that Cu(1) a n d a radical cation derivedfrom a-aminoacetophenone position by NMR. as the dopamine @-monooxygenase inhibitory species. RESULTS

a

MATERIALS AND METHODS

All chemicals were reagent grade unless otherwise noted. Dopamine. HCl, tyramine. HCI, norepinephrine. HCl, and disodium fumarate were from Sigma. Catalase was from Boehringer and ascorbic acid was from Gallard-Schlesinger. Superoxide dismutase was from Worthington. p-Hydroxyphenethylamine.HCl was purchased from ICN or prepared as described below. a-Aminoacetophenone .HCI was from Aldrich and was recrystallized from ethanol-ether prior to use. P-Aminopropiophenone. HCl was prepared according to the method of Hale and Britton (22). Sodium borodeuteride and lithium aluminum deuteride (both 98 atom % D) were purchased from Aldrich. DE52 ion exchange resin was from Whatman and Sephadex G-25 and concanavalin A-Sepharose were from Pharmacia. Methylmannoside was purchased from Calbiochem. Absorbance spectroscopic determinations were performed on a Cary 118B, fluorescence assays assays on a Yellow Springs Instrument polarographic oxygen electrode. HPLC' was performed with a Beckman Model 332 gradient liquid chromatographic system equipped with a Model 155 variable wavelength detector. Mass spectral analyses were obtained on an A. E. I. Model 12 mass spectrometer. Dopamine 0-monooxygenase was prepared and assayed as previously described (13, 21). Synthesis of (R,S)-2-Pheny1-2-hydroxy-[2"80]ethylamine.HC1 (p[lRO] Hydroxyphenethylamine . HC1)- a - Aminoacetophenone . HCI (17 mg, 0.1 mmol) was equilibrated at pH 1.2 in 0.2 ml ofH2180(54% enriched in oxygen-18) containing 1 mM CuClz for 24 h, 35 "C. Labeled water was removed by bulb-to-bulb distillation andthe resulting solid was redissolved in 0.5 ml of Nz-saturated water. This material was immediately added, in a dropwise fashion, to a solution of NaBH4 (30 mg, 0.8 mmol) in IO miof H 2 0 (final pH 8). After 15 min, 0.35 ml of 2 N HC1 was added to the reaction mixture. The total volume of the sample was reduced to 0.35 ml and the@-hydroxy product was purified by HPLC. P-Hydroxyphenethylamine was derivatized by pentafluoropropionic anhydride, followed by mass spectral analysis as previously described (21). The oxygen-18 content of product was calculated from fragmentation peaks-at m/e 253: (M+ 2)/[M + (M + 2)] = 31.4%.

' The abbreviation used is: HPLC, high pressure liquid chromatography.

Dehydration of /I-Hydroxyphenethylamine-derivedgemDiol-The ability of @-hydroxyphenethylamine to generate enzyme-bound a-aminoacetophenone via dehydration of the gem-diol product at the enzyme active site was assessed using @-hydroxyphenethylamine, 31.4% enriched i n "0. The hydroxylation of "0-substrate will yield ['60,'80]gem-diol; if t h e subsequent dehydration of enzyme-bound gem-diol occurs on the enzyme surface, a-aminoacetophenone is expected to be produced with preferential/complete retention or loss of l80 label (Scheme 1). Conversely, dehydration of the gem-diol a random loss of either H,160 after dissociation would lead to or Hz''0, producing a-aminoacetophenone with one-half the original "0 enrichment. Thus, the isotopically labeled substrate was incubated with dopamine P-monooxygenase, and the product a-aminoacetophenone was trapped by sodium borodeuteride reduction. Following HPLC purification, the content of the product-derived @-hydroxyphenethylamine was 14.4 & 1.4% as determined by mass spectrometry (Table

180 , 0%

"0.31 %

" 0 ,I6 %

SCHEME1. Predicted oxygen-18 enrichment of a-aminoacetophenone produced via gem-diol dehydration in solution versus at the enzyme active site. a-Aminoacetophenone enrichments based upon a 31.4% enrichment in P-['80]hydroxyphenethylamine substrate.

7774

Inactivation of Dopamine P-Monooxygenme by p-Chlorophenethylamine 0

&NH3 2H

Product No BZH.

Substrate

A(m+3)

A

(m*I)+A(m+3)

(m+2) m+ (m +2)

&NH3

Product

' ' 0 enrichment

Substrate IBO enrichment

SCHEME 2. Reductive trapping of a-aminoacetophenone derived from gem-diol using sodium borodeuteride provides a means of differentiating starting B-hydroxyphenethylamine from trapped aaminoacetophenone. 0, oxygen-18; 8, the isotopic composition of product. TABLEI Oxygen-18 enrichmentof P-hydroxyphenethylamine obtained by reductive (sodiumborodeuteride) trapping of a-aminoacetophenone produced by dopamine P-monooxygenase-catalyzed hydroxylation of 0-hydroxyphenethylamine The incubation conditions (1-ml total volume) for A were 20 mM potassium phosphate (pH 6), 10 mM fumarate, 2 p~ CuC12, 50 pg ml" catalase, 1 mM @-[180]hydroxyphenethylamine.HCl (31.4% enriched in oxygen-18), 10 mM ascorbate and0.2 mM 0,at 37 "C for 15 min. The dopamine 0-monooxygenase concentration was 100 pg ml-l. Incubation B was identical except for 1 p~ CuCl, and 0.5 mM 0,. After the 15-min incubation, 200 pl of 0.4 M potassium phosphate, pH 7.1, was added, immediately followed by 20 p1 of 1.25 M sodium borodeuteride in 0.01 N NaOH. After 15 min, the reaction mixture was adjusted to pH 6 by the addition of 0.4 M potassium phosphate and the protein was removed by membrane filtration (Centriflo cone). The filtrate was concentrated in uacuo and purified by HPLC. The purification employed an Ultrasphere CIS column (4.6 X 250 mm) with 30% methanol in 0.04 M ammonium acetate as the mobile phase. The isolated P-hydroxyphenethylamine was derivatized by heating with pentafluoropropionyl anhydrideat 70 "C for 45 min and analyzed by mass spectrometry (21). The mass fragment at m / e 253 was monitored because of its intensity; in all cases it possessed the same isotopic profile a the molecular ion at m / e 429. Incubation

A B Average

Conversion"

Product "0 enrichmentb

%

%

20.2 11.7

15.7 13.0 14.4

1.4

"Calculated from [A(M + l)]/[M + A(M + l)],where A(M + 1) equals (M + 1) corrected for natural abundance and I 7 O content in the enriched starting ~-[180]hydr~xyphenethylamine. It should be noted that only the S-enantiomer will function as a substrate and therefore the "true" percentage conversion values are twice those presented.

* These values represent calculation of the product-derived P-hydroxyphenethylamine "0 enrichment using the basicexpression, product l80enrichment = [A(M + 3)]/[A(M + 1) + A(M + 3)]. A(M + 3) equals (M + 3) corrected forthe natural abundance contribution of (M + 2), together with a small empirical contribution to (M + 3) in the starting material (0.65%of M). Control experiments indicated a maximum loss of 20% of the "0 label to solvent during the workUP.

I). The use of sodium borodeuteride allowed for convenient differentiation between startingP-hydroxyphenethylamine and thereductively trapped product (Scheme 2). These results support release of the gem-diol product produced via P-hydroxyphenethylamine hydroxylation prior to dehydration to a-aminoacetophenone, and are consistent with the inability of P-hydroxyphenethylamine to inactivate dopamine P-monooxygenase. Initial Rate Studies of P-Hydroxyphenethylamine Hydroxylation-The dopamine P-monooxygenase-catalyzed hydroxylation of P-['H]- and p-[2H]hydr~xyphenethylaminewas studied as a function of oxygen and amine concentration in an effort to gain insight into the kinetic mechanism. The initial rate of hydroxylation was determined by monitoring oxygen uptake and the results are graphically presented in

a Fig. 1, A and B. Upon examination, it is apparent that quantitative determination of the kinetic parameters is severely hampered by the high K , values for both P-hydroxyphenethylamine (-50 KIM) and oxygen (-0.8 mM). However, the data do provide semiquantitative estimatesof these values and are thereforeuseful in ascertaining the magnitudeof the primary isotope effect on V,,,. As shown in Fig. IC, a replot of the intercept values obtained from Fig. 1, A and B, demonstrates the nearly superimposable natureof the intercepts derived from the @-['HIhydroxy and p-['H] hydroxyphenethylamine primary plots. Therefore, as a first approximation, the apparent isotope effects at the various oxygen concentrations were averaged to obtain the value 1.0 f 0.3 as an estimate of DVmer Thisresult is analogous tothe isotopeeffect previously observed for dopamine hydroxylation under identical experimental conditions, Dk.t= 1.2 (13), and supports product release as the major rate-limiting step for P-hydroxyphenethylamine turnover. Initial Rate Studiesof p-Chlorophenethylamine Hydroxylation-As with P-hydroxyphenethylamine,the initial rate of dopamine P-monooxygenase-catalyzed hydroxylation of P['HI- and P-[2H]chlorophenethylamine was studied asa function of oxygen and amine concentration. Replots of intercepts derived from initial rate data are presented in Fig. 2. The intercept values indicate a large primary isotopeeffect, DVmax(Dkce.t) = 6.1 f 1.2, in marked contrast to the results obtained with theP-hydroxy substrate. This observationof a substantial isotope effect supports major differences in ratedetermining steps for the dopamine P-monooxygenase-catalyzed hydroxylation of @-chloro- andp-hydroxyphenethylamine. We conclude that, for P-chlorophenethylamine, C-H bond cleavage is a significant rate-limiting step. Isotope Effect on @-Chlorophenethylamine Inactivation of Dopamine p-Monooxygenase-Preincubation of dopamine Pmonooxygenase with P-chlorophenethylamine leads to timedependent enzyme inactivation (21). The inactivation occurs concomitant with turnover and appears mechanism-based. Further information regarding the natureof the inactivating species was sought using deuterium isotope effects as a tool to elucidate the kinetic mechanism. The isotopeeffect on inactivation was assessed by preincubation of dopamine Pmonooxygenase with P-chlorophenethylamine containing protium or deuterium at the P-carbon. The results are presented in Fig. 3. The protiated substrateproduced an inactis-', consistent with that vation rate, kinact= 1.1f 0.01 X IOM4 previously reported (21). The deuterated substrate inactivated dopamine P-monooxygenase at a much slower rate, and required amine concentrations of greater than 10 mM in order to show inactivation rates distinguishablefrom controls: since saturation of inactivation was apparent at thehigher concentrations (i.e. 30 and 60 mM), we obtain kinact= 3.0 f 0.3 X s-l as the meanof the observed inactivation rates. These results indicatea significant primary deuteriumisotope effect onactivation, Dkinact= 3.7 f 0.4, implicating C-H bond cleavage in the inactivationprocess. a-Aminoacetophenone As Exogenous Electron Donor-The

Inactivation of Dopamine p-Monooxygenase 0-Chlorophenethylamine by

7775

05

A

1

1

,

1

2

4

6

e

C

0

0

I / B - ['HI-OH (mM)-l l / ~ - [ 2 H ] - O H (mM)-l I I O 2 t mMI" FIG. 1. Lineweaver-Burk plots of initial rates as a function of B-['Hlhydroxyphenethylamine (A) and ,9-[*H]hydroxyphenethylamine( B ) .Rates were determined a t 35 "C by monitoring oxygen uptake using a polarographic oxygen electrode (13,21). Reaction mixtures contained 100 mM potassium phosphate (pH 6), 10 mM fumarate, 10 mM ascorbate, 40 pg ml" catalase, 5 to 30 mM amine, 1 p M CuClZ, and 10 pg ml-' dopamine Pmonooxygenase. Oxygen concentrations were 0.12 mM (a), 0.2 mM (X), 0.41 mM (O), and 0.8 mM (+). Data were fit by nonlinear least squares analysis(13) with weighting factors set to unity. Replots of intercept values from A , 0,and B, A, are given in C. The data inC indicate nearly identical values for P-['H]- and P-[*H]hydroxyphenethylamine; a n estimate of the isotope effect, D$.t various oxygen concentrations.

= 1.0 & 0.3, was obtained as theaverage of the isotope effects at the

I

/

8

L

L

-

0

0

1

2

3

I IO2 ( m MI"

4

5

005

0.1

0 15

I/p-Cl (mM1"

FIG. 2. Replots of intercept values derived from initial rate data for 8-['H]chlorophenethylamine (0) and 8-[2H]chlorophenethylamine (A). Replots as a function of oxygen concentrations, 0.25 to 0.7 mM, and amine concentration, 6 to 30 mM, are shown in A and B, respectively. Rates were determined a t 35 "C by measuring oxygen uptake using a polarographic oxygen electrode (13, 21). Reaction mixtures contained 100 mM potassium phosphate (pH 6 ) , 10 mM fumarate, 10 mM ascorbate, 40 pg ml" catalase, 0.2 to 0.7 mM oxygen, 6 to 30 mM amine, 1 to 2 p M Cucl2, and10-16.5 pg ml" dopamine B-monooxygenase. The higher dopamine 8-monooxygenase and CuC12 concentrations were requiredfor measurement of the substantially reduced rate with ~-[ZH]chlorophenethylamine. We have shownthat isotope effectsare not altered by these concentration changes (data not shown). Data were fit by nonlinear least squares analysis (13),employing weighting factorsgenerated from the analysis of primary plots. Comparison of intercept values in A and B indicates a large primary isotope effect, DV,,,a. (Dk,,) = 6.2 k 1.2. A control experiment, performed by mixing P-['H]- and P-[zH]chlorophenethylamine, verified that deuterated substrate did not contain trace, inhibitory impurities.

FIG. 3. Time-dependent inactivation of dopamine 8-monooxygenaseby &['HI (0) and 8-[*H]chlorophenethylarnine (A). Dopamine 8-monooxygenase (60 fig ml-') was preincubated under conditions of 100 mM potassium phosphate (pH 6), 10 mM fumarate, 10 mM ascorbate, 0.2 mM oxygen, 2 p M Cuc12, and 100 pg ml" catalase a t 35 "C. At various time intervals, a 40-pl aliquot of the preincubation mixture was diluted into 200 pl of assay mixture such that final conditions were 100 mM potassium phosphate (pH 6), 10 mM fumarate, 10 mM ascorbate, 17 mM KCl, 1 mM dopamine, 1 PM CuC12,and 40 pg ml" catalase. Following incubation for 3 min a t 37 "C, the reaction was quenched by the addition of 240 pl of 0.1 N perchloric acid. The product, norepinephrine, was assayed by fluorescence (21). The very slow inactivation rate by P-[2H]chlorophenethylamine necessitated the use of high amine concentrations (210mM) in order to obtain ratessignificantly greater than controls. Since the inactivation appeared saturateda t these high concentrations, kinseta t infinite amine was calculated as themean of three values: kin, = 3.0 -+ 0.3 X s-', leading to Dkinact= 3.7 0.4.

Inactivation of Dopamine P-Monooxygenase by P-Chlorophenethylamine

I / A A P (mM)"

FIG. 4. a-Aminoacetophenone ( A A P ) as electron donor in the dopamine 8-monooxygenase-catalyzedhydroxylation of tyramine. The assay conditions were 100 mM potassium phosphate (pH 6), 3 to 30 mM a-aminoacetophenone, 10 mM fumarate, 10 mM I 2 3 4 tyramine, 40 pg ml-' catalase, 1 p M CuC12,and 15 pg ml" dopamine P-monooxygenase. Therate of hydroxylation was determined by Time of Preincubation ( h ) measuring oxygen uptake. The additionof superoxide dismutase (100 FIG. 5. Oxygen requirement for 8-chlorophenethylamine pg m1-I) had noeffect on the hydroxylation rate. No detectable uptake inactivation under nitrogen (0) or air (W) and a-aminoacetoof oxygen above control levels was observedin the absence of tyramine phenone inactivation under nitrogen (0)or air (0).For 8or enzyme. Data were fit by nonlinear least squares analysis(13). chlorophenethylamine inactivation, enzyme (60 pg ml-I) was preincubated in 100 mM potassium phosphate (pH6), 10 mM fumarate, 10 proposed intermediacy of enzyme-bound a-aminoacetophe- mM ascorbate, 1 p M CucIZ, 10 mM 8-chlorophenethylamine, and 40 none in the inactivation of dopamine @-monooxygenaseby @- pg ml" catalase a t 35 "C. For a-aminoacetophenone inactivation, chlorophenethylamine posed the question as to the natureof enzyme (30 pg ml-') was preincubated in 100 mM potassium phoschemical intermediates leading to inactivation. The structuralphate (pH 6), 10 mM fumarate, 5 yM Cucl2, 20 mM a-aminoacetophenone, and 50 pg ml" catalase a t 35 "C. At various time intervals, similarity of a-aminoacetophenone to ascorbate, particularly a 40-pl aliquot wasremoved and diluted into 200 p1of an assay when in its enol form,led to an examination of a-aminoace- mixture such that final concentrations were 100 mM potassium phostophenone as a potential exogenous electron donor in the phate (pH 6), 10 mM fumarate, 10 mM ascorbate, 17 mM KC1, 1 mM dopamine /3-monooxygenase-catalyzed hydroxylation of do- dopamine, 1 p~ CuCI2, and 40 pg ml" catalase. Incubation for 3 min pamine. The results, shown inFig. 4, indicate that a-amino- was followed by quenching with 240 pl of 0.1 N perchloric acid. The acetophenone can support the hydroxylation of tyramine with product, norepinephrine, was assayed by fluorescence (21).

kc,, (0.2 mM oxygen) = 0.20 & 0.01 s" and an apparentK,,, of 20 f 2 mM. The failure of added superoxide dismutase to affect the hydroxylation rate rules out a role for solutiongenerated superoxide as the reducingspecies (27). As a result of the ability of a-aminoacetophenone to act as an electron donor, together with a demonstrated role for ascorbate as a one-electron donor to dopamine 6-monooxygenase (9, lo), we suggest anintramolecular redoxreaction togeneratethe radical cation,

dressed were the possible role of oxygen and theinvolvement of theenaminetautomer of a-aminoacetophenoneinthe inactivation process. Oxygen was required for the time-dependentinactivation by @-chlorophenethylamine (Fig. 5). Preincubation of enzyme with @-chlorophenethylamine in the absence of oxygen ( N p atmosphere) produced no loss of dopamine @-monooxygenase activity, consistent with turnover as a prerequisite for inhibition. As shown in Fig. 5, a-aminoacetophenone was alsofoundto behighly dependenton oxygen, and only with fairly rigorous exclusionof oxygen was inactivation prevented. The possibility of redox chemistry requiring the participa3 tion of anamino group andketonethroughanenamine as apossible dopamine 0-monooxygenase-inactivatingspe- structure was examined. Although it seemedpromising to since if enzyme-bound 2, species 3 would be study a-[2H2]@-chlorophenethylamine, cies. Incontrasttoenzyme-bound capable of covalent modification and, hence, irreversible in- a-aminoacetophenone must enolize to produce inactivation a-dideuteration could perturb either the rateof formation or activation of enzyme. Inactivation of Dopamine P-Monooxygenme by a-Aminoace- the position of equilibrium toward enamine, noisotope effect was observed when a-[2H2]/3-chlorophenethylaminewas tophenone-The implication that a-aminoacetophenone /3-monooxygenase (datanot bound to dopamineP-monooxygenase is a n obligate interme- preincubatedwithdopamine diatein enzyme inactivation by P-chlorophenethylamine shown). In general, the absenceof an isotope effect in an enzymatic prompted a more detailed investigation of a-aminoacetophenone inactivation in the absence of ascorbate. Two aspectsof reaction does not distinguish chemical mechanisms, since the a-aminoacetophenone inactivation which had not been ad- step under investigation may not be kinetically significant.

Inactivation of Dopamine P-Monooxygenme We therefore examined 0-aminopropriophenone' zyme inactivator:

7777

by Whlorophenethylamine

as an en-

0

Q-I!CH2C"JW 4

If the enamine structureis required for a-aminoacetophenone inactivation, insulation of the amino group from ketone as in the homolog, 4, should abolish the observed inactivation. Consistent with this prediction, @-aminopropriophenone produced no significant inactivationof dopamine P-monooxygenase (Fig. 6). DISCUSSION

In the present report, we have investigated the apparent dichotomy exhibited by P-chloro- and P-hydroxyphenethylamine with regard to dopamine 0-monooxygenase inactivation. As previously reported, whereas both P-chloro- and 0hydroxyphenethylamine are hydroxylated at similar ratesand yield a-aminoacetophenone as a common ultimate product, only the former produces time-dependent enzyme inactivation (21). The inactivationby P-chlorophenethylamine appears to be mechanism-based in that deletion of either reductant (21) or oxygen (Fig. 5) preventsactivity loss. The inactivation occurs at a slow rate, kinact= 0.94-1.1 x s-l (Ref. 21 and Fig. 2) relative to turnover, kc,, = 1.1-3.7 s-' (Ref. 21 and Fig. 2; 0.2 mM oxygen), leading to anirreversible loss of enzymatic activity. Based upon the inability of a-aminoacetophenone to produce inactivation under turnover conditions and its production from both 0-hydroxy- and @-chlorophenethylamine, it appeared unlikely as the inactivating species. However, in the absence of ascorbate as reductant, conditions under which dopamine P-monooxygenase exists largely in the Cu(1I) form ( 7 ) ,a-aminoacetophenone proved to be an enzyme inactivator, kinact 1x s-l (21). This finding raised the possibility of a differential susceptibility of dopamine 0-monooxygenase to a-aminoacetophenoneinactivationdependent upon the enzyme's redox state, and led to the proposed formation of bound ketone, 2, in the course of P-chlorophenethylamine inactivation. A major difference ina-aminoacetophenoneproduction from 0-hydroxy- versus P-chlorophenethylamine is the predicted stability of their hydroxylated intermediates. Specifically, the gem-diol derived from 0-hydroxyphenethylamine is predicted to be considerably more stable than the chlorohydrinobtained from 0-chlorophenethylamine. This obvious disparity suggested dehydrohalogenation of the chlorohydrin prior to dissociation as a means of generating a-aminoacetophenone at the activesite of dopamine-P-monooxygenase while in the susceptible E-Cu(I1) oxidation state (cf. Scheme 3). The more stable 0-hydroxy-derived gem-diol is predicated to dissociate priorto dehydration, and is thus unable to generate a-aminoacetophenone at the active site. To test this hypothesis, P- ['80]hydroxyphenethylamine was incubated with dopamine P-monooxygenase, and the product a-aminoacetophenone was trappedwith sodium borodeuteride. As shown inTable I, random loss of the "0 label supports dissociation prior to dehydration, and is consistent with the predicted inability of 0-hydroxyphenethylamine to inactivate dopamine 0-monooxygenase via the generation of active site a-aminoacetophenone.

-

I

2

3

4

Time of Preincubation ( h )

FIG. 6. Effect of preincubation of j3-aminopropiophenoneon

dopamine j3-monooxygenase activity. Dopamine P-monooxygenase (30 pg ml-') was preincubated in 100 mM potassium phosphate (pH 6), 10 mM fumarate, 5 p~ Cucl2, and 5 Q p g ml-' catalase at 35 "c either in the absence of amine ( X ) or in the presence of 20 mM paminopropiophenone (A) or 20 mM a-aminoacetophenone (0).At various time intervals, 40-pl aliquots were removed and assayed by fluorescence for dopamine P-monooxygenase as described in Figs. 3 and 5.

SCHEME 3. Proposed mechanism for the interaction of j3chlorophenethylamine with dopamine 8-monooxygenase. As indicated, a radicalcationderivedfromenzyme-boundketone is postulated as the species leading to enzyme inactivation.

Additional information regarding differences in the dopamine P-monooxygenase-catalyzed hydroxylation of @-chloroand P-hydroxyphenethylamine was obtained by analysis of primary deuteriumisotope effects. Previous studies of deuterium isotope effects in dopamine hydroxylation (13) indicate release of product norepinephrine as the major rate-limiting step, Dkcat = 1.2 (pH 6.0, 10 mM fumarate). The deuterium isotope effect observed for P-hydroxyphenethylamine is similar (Fig. 1)with an estimated Dkcat= 1.0 k 0.3. Thus, despite large increases in K , values with 0-hydroxyphenethylamine, dopamine and 0-hydroxyphenethylamine are characterized by Although we have not looked for bindingof P-aminopropriophen- similar values for kcatand Dkeat (Table 11). Examination of the initial rate data for 0-chlorophenethylamine reveals a subone, both Creveling et al. (28) and May et al. (16) report that phenpropylamine is a substrate for dopamine P-monooxygenase. stantial kinetic isotope effect, Dkc,, = 6.2 1.2. This result

*

7778

Inactivation of Dopamine P-Monooxygenaseby P-Chlorophenethylamine TABLE I1 Comparison of kinetic parameters for the hydroxylationof dopamine, p-hydroxy-, and /3-chlorophenethylamine by dopamine @-monooxygenme Calculated using the expressions Dk,at = (Dk + k5/k7)/(1 + k&7) and kcat= k5k7/(k5+ k7) assuming a value of Dk = 9.4 (24). The constants k, and k , refer to Scheme 3. S -1

Dopamine"

13

mM

0.5

S-1

1.2

550

S"

13

HoYNH2 HO

(R,S)-p-Hydroxyphenethyl-

1.39

1.50

4.3

2980

13

15

6.1

21

-39

amineb

LJ c1

(R,S)-@-Chlorophenethylamine'

___

....

"~

31

..

From Ref. 13. * From results presented in Fig. 1. From results presented in Fig. 2.

indicates C-H. bond cleavage is significantly rate limiting in However, this mechanism, which involves the partitioning of P-chlorophenethylamine hydroxylation. As suggested earlier a common intermediate between inactive enzyme, kin,,,, and from data at a single oxygen concentration (21), K,,, values product, $, predicts identical isotope effects on kinactand k,,,. for P-chlorophenethylamine hydroxylation are reduced rela- For this reason, as well as a number of key factors outlined tive to P-hydroxyphenethylamine (Table 11). However, the below, we consider thealternatemechanism in which a large isotope effect for 0-chloro hydroxylation indicates this aminoacetophenone is the inactivating species (Scheme 3). substrate is, in fact, farless reactive with dopamine 0-hydrox- Thismechanismis favoredfor the following reasons: (i) ylase than P-hydroxyphenethylamine. Employing the intrin- enzyme-bound chlorohydrin is predicted tobe lessstable than sic isotope effectof 9.4 recently determinedfor dopamine (24), enzyme-boundgem-diol,&/k7 (P-chloro)>> &/k7 (@-hydroxy), we estimaterateconstants of 2 1 and 31 s" for 6-chloro in contrast to theunlikely constraint required by the mechahydroxylation and product release (k5 and k7, Scheme 3). By nism inEquation 3 that kinact/$ (P-chloro) >> kinact/kp(panalogy to dopamine (13), the magnitude of Dkc,t for 8-hy- hydroxy); (ii) enzyme-bound gem-diol is concluded to dissodroxyphenethylamine implies that C-H bond cleavage is at ciate prior to dehydration to a-aminoacetophenone (Table I); least 25-fold faster than productrelease, kg 2 25k7 2 980 s-'. (iii) dopamine /3-monooxygenase is inactivated by a-aminoThe above estimates suggest a similar product dissociation acetophenone in the absence of ascorbate (Ref. 21 and Figs. serve as electron rate, k7(p-chloro) = 31 s" uersus k7(P-hydroxy) 39 s-', but 5 and 6); and (iv) a-aminoacetophenone can dramatically different rates of hydroxylation, k5(P-chloro) = donor in the dopamine(3-monooxygenase reaction (Fig. 4). The proposed intermediacy of active site-generated a-ami21 s" uersus k,(P-hydroxy) 2 980 s" (Table 11). These differences may be indicative of C-H bond cleavage to generate noacetophenone, 2, in P-chlorophenethylamine inactivation either a radical or carbocation intermediate, since electronic- raised the question of the chemistry of enzyme inactivation ally the hydroxyl substituent is expected to stabilize such (21). The structural similarity of the enamineof a-aminoacetransition states/intermediates while the chloro substituent tophenone to ascorbate, coupled with its known propensity to would oxidatively dimerize in alkaline solution (25), suggested an A mechanistically important finding is a substantial pri- internal redox reaction as a possible pathway to inactivation. This possibility is supported by the ability of a-aminoacetomarydeuterium isotopeeffect onP-chlorophenethylamine inactivation of dopamine 0-monoxygenase, Dkinaet= 3.7 f 0.4 phenone to substitute for ascorbate as the exogenous reduc(Fig. 3), implicatingC-H bond cleavage as a prerequisite for tant in substratehydroxylation (Fig. 4). Tyramine was chosen inactivation. Although Dkinactwas only determined at a single as substrate for the experiment in Fig. 4 because of its rapid Dkinact< "kcat. The rate of hydroxylation by dopamine 0-monooxygenase, and its oxygen concentration,itappearsthat distinction between the kinetic propertiesof 8-chloro- andP- inability to function as reductant (in contrast to dopamine hydroxyphenethylamine (Table11) raised thepossibility of an which turns over in the absence of added reductant; Ref. 26). inactivation mechanism involving the formation of a radical The effectiveness of bound a-aminoacetophenone in substrate hydroxylation is roughly 10-fold less than ferrocyanide, with (or carbocation) adjacent to achlorogroup, since such an intermediate is expected to be less stable (and thereforemore an observed apparent K , of 20 mM and kc,, (0.2 mM oxygen) of 0.20 s-'. The possibility that the actual reducing species reactive) than that of the correspondinghydroxy compound was solution-generated superoxide (27) was ruled out by the failure of added superoxide dismutase to inhibit the enzymatic reaction. Thus, the redox reaction appears to involve a direct interaction with the enzyme, presumably at the active site. Interestingly, thehigh apparent K , of 20 mM is similar to the EQUATION 3 reported Kinactof approximately 50 mM (21), reflective of a relatively low affinity of a-aminoacetophenone under condi3Examination of u+ or u values indicates: u+(OH) = -0.92, u(OH) = -0.37, o'(C1) = +0.11, a(C1) = 0.23 (29). Both carbocations, tions of enzyme turnover and inactivation. Regarding the and, in general,radicalreactions are characterized by negative p species of a-aminoacetophenone participating in theseprocvalues of variable magnitude (30). esses, the involvement of an enamine structure is supported

- u

vgenase P-Chlorophenethylamine by Inactivation of Dopamine @-Monoox:

7779

6. Blumberg, W. E., Goldstein, M., Lauber, E., and Peisach, J. by analogy to ascorbate, and the demonstrated inertness of (1965) Biochim. Biophys. Acta 9 9 , 187-190 dopamine 0-monooxygenase towardthea-aminoacetophe7. Walker, G. A., Kon, H., and Lovenberg, W. (1977) Biochim. none homolog, 4 (Fig. 6 ) . Biophys. Acta 482,309-322 Although Scheme 3 is based, inpart,onthe observed 8. Terland, O., and Flatmark, T. (1975) FEBS Lett. 59,52-56 inactivation of dopamine P-monooxygenase by a-aminoace9. Skotland, T., and Ljones, T. (1980) Biochim. Biophys. Acta 6 3 0 , tophenone, it remains to be shown that P-chlorophenethyla30-35 mine and a-aminoacetophenone inactivate enzymevia the 10. Diliberto, E. J., Jr., and Allen, P. L. (1981) J. Biol. Chem. 2 5 6 , 3385-3393 same intermediate. Of particular note is the autooxidationof a-aminoacetophenone in solution to generate hydrogen per- 11. Klinman, J. P., Krueger, M., Brenner, M., and Edmondson, D. F. (1984) J. Biol. Chem. 2 5 9 , 3399-3402 ~ x i d e Thus, .~ it is possible that a-aminoacetophenone func- 12. Klinman, J . P., Humphries, H., and Voet, J. G. (1980) J. Biol. tions in a dual fashion: first, to reduce E-Cu(I1) to E-Cu(1); Chem. 2 5 5 , 11648-11651 and second, toproduce hydrogen peroxide, which inactivates 13. Ahn, N., and Klinman, J. P. (1983) Biochemistry 22,3096-3106 dopamine P-monooxygenase in a very rapid, possibly diffu- 14. May, S. W., and Phillips, R. S. (1980) J. Am. Chem. SOC.1 0 2 , 5981-5983 sion-controlled, reaction with the E-Cu(1) form of e n ~ y m e . ~ In this instance, the demonstrated requirement for oxygen in 15. Baldoni, J. M., and Villafranca, J . J. (1980) J. Biol. Chem. 255, 8987-8990 a-aminoacetophenone inactivation (Fig. 5 ) would be to pro- 16. May, W. S., Mueller, P. W., Padgette, S. R., Herman, H. H., and duce hydrogen peroxide. By contrast, in Scheme3, the role of Phillips, R. S. (1983) Biochem. Biophys. Res. Commun. 1 1 0 , oxygen is ascribed to the maintenance of enzyme in an oxi161-168 dized, ketone-sensitive form. An important piece of informa- 17. Levin, E. Y., Levenberg, B., and Kaufman, S. (1960) J. Biol. Chem. 235,2080-2086 tion, currently under investigation, concerns the stoichiometry of covalent modification of enzyme in the course of inac- 18. Kaufman, S., Bridges, W. F., Eisenberg, F., and Friedman, S. (1962) Biochem. Biophys. Res. Commun. 9 , 497-502 tivation by both P-chlorophenethylamine and a-aminoaceto- 19. Taylor, K. B. (1974) J. Biol. Chem. 2 4 9 , 454-458 phenone. These results should indicate whether inactivation 20. May, S. W., Phillips, R. S., Mueller, P. W., and Herman, H. H. is active-site specific, and further, if the inactivating species (1981) J. Biol. Chem. 256,2258-2261 with a-aminoacetophenone is an enzyme-bound radical ca- 21. Klinman, J. P., and Krueger, M. (1982) Biochemistry 21,67-75 22. Hale, W. J., and Britton, E. C. (1919) J. Am. Chem. SOC.4 1 , tion, 3,versus an activated formof oxygen. 841-847 23. Barnett, J., Dupre, D. J., Halloway, B. J., and Robinson, F. A. REFERENCES (1944) J. Chem. SOC.94-96 1. Winkler, H. (1976) Neuroscience 1 , 65-80 24. Miller, S. M., and Klinman, J. P. (1983) Biochemistry 22,30913096 2. Nagatsu, T. (1972) Trends Biochem. Sci. 2 , 217-219 3. Skotland, T.,and Ljones, T. (1979) Znorg. Perspect. Biol. Med. 25. Vinot, N., andPinson, J. (1968) Bull. SOC.Chim. France 1 2 , 151-180 4970-4974 4. Rosenberg, R. C., and Lovenberg, W. (1980) in Essays in Neuro- 26. Craine, J. E., Daniels, G. H., and Kaufman, S. (1973) J. Biol. chemistry and Neuropharmacology (Youdin, M. B. H., LovenChem. 248,7838-7844 berg, W., Sharman, D. F., and Lagnado, J. R., eds) Vol4, John 27. Henry, J. P., Hirata, F., and Hayaishi, 0.(1978) Biochem. BioWiley & Sons, Ltd., New York phys. Res. Commun. 8 1 , 1091-1099 5. Friedman, S., and Kaufman, S. (1966) J. Biol. Chem. 241,225628. Creveling, C. R., Daly, J. W., Witkap, B., and Udenfriend, S. 2259 (1962) Biochim. Biophys. Acta 6 4 , 125-134 29. Ritchie, C. O., and Sager, W. F. (1964) Prog. Phys. Org. Chem. 2 , 323-400 M. Bossard and J. P. Klinman, unpublished results. Such arapidinactivation process could proceed even in the 30. Pryor, W.A., Lin, J. H., and Henderson, R. W. (1973) J. Am. Chem. SOC.95,6993-6998 presence of high levels of catalase, depending on the magnitude of second order rate constants for the reaction of hydrogen peroxide 31. Ash, D. E., Papadopoulos, N. J., Colombo, G., and Villafranca, J. with catalase uersus the E-Cu(1) form of dopamine 8-monoxygenase. J. (1984) J . Biol. Chem. 259, 3395-3398