In the present study, we have examined the in vitro steady- state kinetic ... adjusted to 5.4 with NaOH) and the pH of the MES buffer adjusted as necessary to ...
Vol. 266, No. 18, Issue of June 25, PP. 11537-11543.1991
THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistryand Molecular Biology, Inc
Printed in U.S.A.
Cooperativity in the Dopamine &Monooxygenase Reaction EVIDENCEFORASCORBATEREGULATIONOFENZYMEACTIVITY* (Received for publication, August 2, 1990)
Leslie C. Stewart$ and Judith P. KlinmanQ From the Departmentof Chemistry, Universityof California, Berkeley, California 94720
The steady-state kinetic behavior of dopamine 8- approximately equal distribution of enzyme activity between monooxygenase (DBM) has beenexaminedovera the two forms (1-4). Ascorbic acid,which is present at a 1000-fold range of ascorbate concentrations. Kinetic concentration of 10-20 mM within adrenal chromaffin granplots exhibit extreme curvature indicative of apparent ules (5-7) and is the most effective i n vitro reductant for negative cooperativity in the interactionof D@Mwith dopamine P-monooxygenase (8), is the presumed i n vivo reascorbate, with a calculated Hill coefficient of 0.15- ductant. 0.30. The observed cooperativity is found to be indeAlthough mechanistic aspects of the chemical reaction catpendent of enzyme concentration and tyramine and alyzed by DPM havebeenstudiedindetail (9-ll), much oxygen concentrations,as well as the pH employed for remains unknown concerning possible means of regulation of the assay. Similar kinetic data have been obtained with enzyme activity within chromaffingranules. In particular, the both soluble and purified membrane-derived forms of enzyme. An investigation of the effect of the anion precise mechanisms by which the membraneous and soluble activator fumarateupon the observed kinetic patternsenzyme forms receive the necessary reducing equivalents for has demonstrated a conversion to a less cooperative catalysis remain unclear. In spite of the relatively high conkinetic pattern at low pH and high concentrations of centration of ascorbate within chromaffin granules, the confumarate. This phenomenon is attributed to an inhibi- centration of intragranular catecholamines is in the range of tory binding of the structurally similar monoanionic -0.5 M (12), necessitating theexistence of a recycling system species of fumarate to the ascorbate reductant site. A for intravesicular ascorbateor some additional source of electrons to maintain DPM activity. Although endogenous catesimple model has been used to assess the change in apparent VmaXand K , parameters with increased as- cholamines themselves are incapable of sustaining elevated corbate concentrations. At all pH values examined, DPM activity (8), evidence has accumulated in favor of an of DBM for intragranular recycling system for ascorbate(13-15). Accordthere is a dramatic decrease in the affinity ascorbate from a K , of -0.05-0.10 mM (ascorbate ing to literature models, semidehydroascorbate generated in concentration < 1 mM) to K , > 10 mM at limiting the course of DPM catalysis is rapidly reduced by cytosolic is a 3- to4-fold ascorbate; at the same time there ascorbate via the cytochrome b ~ , ,presentinthegranule increase in the limiting V,,, value. Several models membrane. More recently, results from this laboratory have have been considered to explain the observed activa- provided evidence that membranous dopamine P-monooxytion of DBM by high levelsof ascorbic acid. genase can be reduced from the extravesicularface (16-18).’ It appears that cytosolic ascorbate may interact with the membraneous enzyme through a membrane-bound mediator, Dopamine P-monooxygenase (DPM)’ catalyzes the conver- suggesting that dual modes exist for reductionof soluble and sion of dopamine to the neurotransmitter and hormone nor- membrane-bound DPM within chromaffin granules. These epinephrine (Equation 1)within the catecholamine secreting findings raise the possibility that the pathway for reduction vesicles (chromaffin granules)of the adrenal medulla, as well of intragranular DPM could be a means of regulation for the as the large dense-cored synaptic vesicles of the sympathetic enzyme i n vivo, and further, that thismay occur via different mechanisms for the membraneous and soluble enzyme spenervous system. cies. Support forapossibleregulatory role for ascorbatehas come from the work of Menniti et al. (20) in which the K, of dopamine P-monooxygenase for reductant ascorbate was deOH termined inwhole chromaffin cells, isolated chromaffin granWithin adrenal chromaffin granules, DPM is known to exist ules, andchromaffin granuleghosts.Significantly, these as both asoluble andmembrane-boundprotein,withan workers observed a K , for ascorbate of 15-17 mM in all three systems, a value substantially higher than a K, of 0.6 mM * This work was supported by Grant GM 25765 from the National reportedearlier for isolated solubleenzyme (21, 22) and Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must subsequently for DPM in intact chromaffin granules (50).3 therefore be hereby marked “advertisement” in accordance with 18 The finding of an elevated K , for ascorbate in chromaffin granule ghosts appears to rule out an interaction of either U.S.C. Section 1734 solely to indicate this fact. $ Present address: Dept. of Biology, University of California San DPM or ascorbate with soluble intragranular components as Diego, La Jolla, CA 92093. § T o whom correspondence and reprint requests should be addressed. I The abbreviations used are: DPM, dopamine 0-monooxygenase; MES, 2-(N-morpholino)ethanesulfonicacid; HPLC, high pressure liquid chromatography.
In accord with an earlier finding (19). Dhariwal et al. (50) attributed the difference between their results and those of Menniti et al. (20) to the use of dopamine, rather than tyramine, as substrate. However, no significant kinetic differences are known to existfor these two substrates (cf. Ref. 11).
11537
‘I
11538
Ascorbate Regulation
of Dopamine P-Monooxygenase
the cause of the large decrease in enzyme affinity for ascorbate. Recently, Huyghe and Klinman (18) have confirmed a high K, for internal ascorbate in chromaffin granuleghosts. In the present study, we have examined thein vitrosteadystate kineticbehavior of both membranous andsoluble DPM over a 1000-fold range of ascorbate concentrations. Within the pH range examined (pH 5.2-6.7), the resulting velocity profiles exhibit extreme nonlinear behavior indicative of apparent negative cooperativity. Numerous control experiments have ruled out the contribution of (i) a slow ascorbate-induced protein conformational change,(ii) a random binding of substrate and oxygen, or (iii) a n association/dissociation of dimeric enzyme subunits as thesource of cooperative behavior with ascorbate. Nonlinear least squares analysis of the experimental data, using the model of Alberty et al. (23) for cooperative substratebinding, has allowed calculation of apparent limiting V,,, and K,,, parameters corresponding to the low and high ascorbate concentrationregions of the experimental kinetic curves. Significantly, our results indicate a very large K,,, a t high ascorbate concentrationsof 10-40 mM (membranederived DPM) and 16-50 mM (soluble DPM), in accord with findings of Menniti et al. (20) and Huyghe and Klinman (18) for intravesicular DPM. Possible models for ascorbate regulation of dopamine P-monooxygenase activity within adrenal medulla chromaffin granules are discussed. EXPERIMENTALPROCEDURES
Materials All reagents were analytical gradewheneverpossible and were purchased from the following sources: ascorbicacid (gold label, >99%) and tyramine hydrochloride were from Aldrich; disodium fumarate, dopamine hydrochloride, heptanesulfonic acid, and ascorbate 2-sulfate (dipotassium salt)were from Sigma; MES was from Calbiochem; and catalase was obtained as a crystalline suspension (65,000 units/ mg) from Boehringer Mannheim. Methods Preparation of Soluble Dopamine p-Monooxygenme-Chromatographic purification of soluble DBM from bovine adrenalglands followed previously published procedures (24, 25), utilizing a lysate of either density gradient purified chromaffin granules, acrude isolate of chromaffingranulesor a direct lysis of homogenized adrenal medullae (26). Enzyme was stored in 10 mM MES buffer, pH 6.5, at -78 "C. As determined by steady-state rate of oxygen consumption, the specific activity of isolated soluble dopamine P-monooxygenase is consistently found to be within 10% of 6 pmol/min.mg utilizing a standard assay system of 1 mM dopamine hydrochloride, 0.21 mM O,, 10 mM ascorbate, 10 mM fumarate, 100 pg/ml catalase, and 100 mM potassium phosphate, pH 6.0,35"C (26). Enzymepurified in thisway shows the typical two band pattern (at 72 and 75 kDa) under conditions of sodium dodecyl sulfate-gel electrophoresis (27). Preparation of Membranous Dopamine 0-Monooxygenme-Membraneous DBM was purified from isolated chromaffin granules as described (27). Preparation of Lysed Washed Chromaffin Granule MembranesChromaffin granule membranes were prepared by lysis of isolated chromaffin granules in dilute (5mM) MES buffer containing 100 pg/ ml catalase, followed by several cycles of freezing and thawing in the presence of lysis buffer. Concentration of total membrane protein was determined in 0.5% ocytl glucoside by the method of Bradford (28) using bovine serum albumin asa standard. Assay of Soluble and Membranous DPM Actiuity-Dopamine pmonooxygenaseactivity was assayed as the initial rate of oxygen consumption (at 2-5 pg/ml protein concentration) using a Yellow Springs model 53 polarographic oxygen electrode or as the rate of octopamine formation usinga discontinuous HPLC/optical detection assay. The latter assay was used a t very low (0.1 pg/ml) and high (1 mg/ml)concentrations ofDBM. Inboth assays, stockascorbate solutions of 1-200 mM were prepared (pH of 100-200 mM stocks adjusted to 5.4 with NaOH) and the pHof the MES buffer adjusted as necessary to maintain a constant pH (at a constant final buffer concentration of 50 mM MES); all reactions contained 100 &ml
catalase and 2 p M CuS04 andwere initiated by the addition of DBM unless otherwise noted. Althoughthe ionic strength of assay solutions increased with increasing ascorbate concentrations, control experiments with added NaCl at low ascorbate indicated similar nonlinear plots;consequentlythe ionic strength was allowed to vary with ascorbate concentrations in subsequent experiments. Exact reaction conditions are noted in figure legends. Where relevant, oxygen concentration was varied by mixing pure oxygen with nitrogen to obtain the desired final concentration. The effects of ascorbate 2-sulfatewere examined at pH6.7 (minus fumarate) in the presence of 10 IIIM tyramine hydrochloride and 1 mM ascorbate. Velocities were determined plus and minus 20 mM ascorbate 2-sulfate using20 mM potassium sulfate as a control. A t a DBM concentration of 0.1 pg/ml, individual reaction mixtures were incubated a t 37 "C for 5-30 min and quenched to a final HCI concentration of 0.3 M. At 1 mg/ml DBM concentration, aliquots (50 pl) of preincubated (37 "C)enzyme stock solutions containing 2 mg/ ml DPM, 100 mM MES, pH5.95, 0.4 mg/ml catalase (because of the short time scale involved in this assay, catalase is not an absolute requirement)and sufficient CuS04toobtain a 2:l copper atom/ subunit ratio (29) were rapidly mixed with aliquots (50 pl) of appropriate preincubated substrate stock solutions containing 30 mM tyramine andtwice the desired ascorbate concentration. Reactionswere allowed to proceed on a time scale of seconds and were quenched to a final HCl concentration of 0.3 M. A t both extremes of protein concentration, octopamine formation was assayed by injection of samples (neutralized with NaHCOx to a final HCOT concentration of40 r n ~ onto ) a standard Altex C-18 reverse phase HPLC column equilibrated in a buffer of 5 mM acetic acid, 6.5% methanol, and 60 p~ heptanesulfonic acid brought to pH 5.8 with 4 M NH40H (26). The octopamine peak elutes sharply at -8 min in thisbuffer system; tyramine elutes asa broad peak at approximately 40 min. A Shimadzu LC-GA/SPD-GA chromatograph/detector and C-R3A integrator system were employed for quantification of octopamine peaks. Computer Fitting of Velocity Datu-Initial velocity data were plotted in a double-reciprocal fashion to obtain estimates of the limiting kinetic parameters V,, K,, V,, and K2 (Equation 2 of text). These estimates were refined according to Equation 2 by use of a nonlinear least squares fitting routine based on the algorithm of Marquardt (30) written by ThomasPollard at the University of California, Berkeley, and run ona LSI-11 computer. RESULTS
Cooperative Kinetic Behavior with Varying Ascorbate Concentration-As seen in Fig. 1, determination of initial velocities withsoluble DPM as a function of ascorbate concentration in the absenceof fumarate gives rise to pronounced curvature, indicative of apparent negative cooperativity. The same pronounced cooperativity is observed using purifiedmembranous enzyme (data not shown (36)) and lysed washed chromaffin granule membranes (Fig. 2) as sources of DPM activity. The degree of curvature, as assessed by calculation of the Hill coefficient ( n H ) is , quite constantover the pHrange examined; values of nH in the absenceof fumarate fall within the range of 0.15-0.30, with some variability among different preparations of enzyme. Ascorbate concentrations corresponding to the minimal Hill coefficient (maximal cooperativity) are in the range of 0.7-1.5 mM. Although the data of Fig. 1 were determined with the nonoxidizable substrate tyramine, no differences have been observed with dopamine as substrate. As noted in thelegend to Fig. 1,identical velocity profiles are obtained upon initiation of reactions directly with DPM or upon preincubation (up to 30 min) of enzyme with thedesired ascorbate concentration and initiation of the reaction with substrate tyramine, indicating thata slow ascorbate-induced protein conformational change (31, 32) is not the cause of nonlinear kinetic curves. As with any observation of kinetic cooperativity, the possibility exists that heterogeneity within the purified enzyme may be contributingartifactuallytothe observed kinetic patterns (33). As discussed (27, 34), there is subunit hetero-
of Dopamine ,&Monooxygenase
Ascorbate Regulation A
351
3
30ti 0 ‘
i1
A: pH 5.2
. I
20
0
60
40
80
100
J
vllascorbatel
B 12
1
‘1
B: pH 6.7
0
I
‘
0
5
15
10
20
30
25
35
V/[o5COIbatel
FIG. 1. Cooperative kinetic behavior as a function of variable ascorbate concentration for soluble dopamine b-monooxof oxygen ygenase. Velocities were measured as the initial rates consumption in the presence (+) or absence (a) of 10 mM fumarate at pH 5.2 ( A ) and pH 6.7 ( B ) ,using 10 mM tyramine hydrochloride and enzyme concentrations of 2 pg/ml, pH 5.2, and 2.5 pg/ml, pH 6.7. Identical velocities were obtained upon initiation of reactions directly with enzyme or upon preincubation ofDUM with ascorbate and initiation with tyramine. The curves drawn (minus fumarate) representthetheoretical fits totheexperimentaldata given the proposed model of Equation 2 (see Tables I, 11, and “Discussion”); lines drawn through 10 mM fumarate data do not represent a theoretical fit.
o i 0
.
, 100
.
~
200
.
, 300
.
, 400
.
11539
terns for both soluble and membrane-derived enzyme forms, as well as for DPM activity in lysed chromaffingranule membranes, would appear torule out a contribution of enzyme microheterogeneity to thecooperative effects. The possible co-elution of damaged monooxygenase during the isolation procedures for soluble and membranousDPM is somewhat more difficult to assess. Columbo et al. (35), in an attempt to address the wide variation in reported specific activities for soluble DPM, have reported that inclusion of catalase in the granulelysis medium protects against enzyme oxidation and results in a final specific activity -1.5 times the value reported by our laboratory (24, 26). However, catalase is routinely included in our granule lysis buffers and the activity and purity of final purifiedenzyme are extremely consistent among the three different lysis procedures employed (c.f. “Methods”). Significantly, freeze-quench kinetic studies from this laboratory (26) indicate that the bulk of enzymic copper in soluble DPM can be reduced by ascorbate in a single exponential process with a limiting rate constant of 250 s” and K,,, = 0.9 mM; only 10% of the enzymic copper could not be reduced at all and was attributed to co-purification of oxidatively inactivated enzyme. Finally, the specific activity of‘ purified membrane-derived dopamineP-monooxygenase (-8.5 pmol/min.mg at pH 5.2) is consistent with the value of 8.2 pmol/min. mg at pH 5.0 reported by Saxena and Fleming (34). Because of previous observations thatDPM exhibits protein concentration-dependent cooperative kinetic behavior with respect to tyramine (36-38) and ascorbate (37, 38), the dependence of the observed curvature of Fig. l (determined a t a concentration of 2-5 pg/ml) on enzyme concentration was examined with soluble enzyme at the extremes of 0.1 pg/ml and 1 mg/ml (Fig. 3); these concentrations are assumed to represent approximatelyexclusive populations of dimeric and tetrameric enzyme,respectively (36). Although the level of tyramine substrate employed (15 mM) and the nature of the assay prohibited determining accuratevelocities above 4 mM ascorbate at 1 mg/ml protein, the dataof Fig. 3 clearly exhibit the same pronounced curvature as seen in Fig. 1, suggesting no relationship between non Michaelis-Menton kinetic behavior and reversible enzyme oligomerization. As a further control to determine whether theuse of nonsaturating levels of oxygen or tyramine over the experimental range of ascorbate concentrationscould give rise to apparent nonlinear kinetic patterns, Eadie-Hofstee profiles were con-
I 500
vl[ascorbate]
FIG. 2. Cooperative effects in lysed washed chromaffin granule membranes. Velocities were determined in 50 mM MES, p H 5.5, over an ascorbate concentration range of 0.05-50 mM in the absence of added fumarate a t a tyramine concentration of 15 mM. Catalase (100 pg/ml) andCuS04 (2 p ~were ) added to assay mixtures, and the concentrationof membrane protein was 80 pglml.
‘1 10
20
30
v/[ascorbate]
FIG. 3. Protein concentration dependence of the observed geneity within both soluble and membranous dopamine mon- cooperative kinetic behavior. As described under“Methods,” ooxygenase, withthemembranous enzyme containing a velocities were measured a t 0.1 pg/ml (m, 0.1-15 mM ascorbate) and higher proportion of the heavier 75-kDa subunit. However, 1 mg/ml (0,0.1-4 mM ascorbate), in the presence of 15 mM tyramine the observationof essentially identical nonlinear kinetic pat- hydrochloride at pH 5.95.
Ascorbate Regulation of Dopamine P-Monooxygenase
11540
structed at varying levels of both tyramine and oxygen. As seen in Fig. 4, the extent of curvature at 0.5 mM oxygen is essentially equivalent to thatobserved at atmospheric oxygen, with identical calculated Hill coefficients at both oxygen concentrations. Similarly, velocity profiles determined at nonsaturatingconcentrations of tyramine (0.5 mM, datanot shown (36)) were found to exhibit similar cooperative behavior to that determined at concentrations of 10-15 mM (Figs. 1, 3, and 4). These findings, which have been confirmed in separatepreparations of soluble enzyme, argue againsta relationship between the order of substrate and oxygen binding and apparentkinetic cooperativity. Effects of Fumarate-DopamineP-monooxygenaseis known to be activated by a wide array of halides and carboxylate-containing anions, of which fumarate is the most effective (21,35,36). Despite the apparenthigh specificity of DPM for activation by fumarate, this compound is not present as a soluble component of adrenal chromaffin granules (36), suggesting an as yet unidentified endogenous activator. Inspection of the monoanions of fumarate and ascorbate, 1 and 2 below, indicates some structural similarity, HO ”
_
\
H
\
c=c.
-0‘
Y=O
CHsOH -0)==(OH
I‘o
2
1
suggesting that ascorbate may activate DPM by binding at the site of fumarate activation and thatfumarate could cause enzyme inhibition through binding at theascorbate reductant site. Consequently, an investigation was undertaken of the possible effects of fumarate upon ascorbate activation, using soluble dopamine P-monooxygenase. As seen in Fig. 1, the presence of 10 mM fumarate has a pronounced pH-dependent effect on the observed curvature in Eadie-Hofstee plots. At pH values from 5.2 to 6.0, the presence of fumarate is inhibitory at low ascorbate concentrations, giving rise to an approximately linear kinetic pattern at pH 5.2. At pH 6.7, however, fumarate is clearly activating enzyme activity over the entire range of ascorbate concentrations (0.05-50 mM). Previous kinetic studies of DPM at high ascorbate concentrations have shown apH-dependentin-
crease in the K,,, for oxygen, which rises from -0.1 mM at pH 6.0 to -0.5 mM at pH7.0.4A detailed analysis of the effect of fumarate on V,,, versus V,,,/K,,, revealed a slight inhibition of VmaX,together with a large activation of V,,,/K,,, (51). We therefore attribute the observed pH-dependent behavior of fumarate at low ascorbate concentrations to two opposing effects, which involve (i) competitive binding of fumarate to the ascorbate reductant site and (ii) a trend away from rate limitation by V,,, toward V,,,/K, with increasing pH. The decrease in fumarate inhibition with rising pH may also reflect its ionization to a dianion (pKl -3.1, pK2 -4.6, (40)), which would not be expected to compete as effectively as its monoanion toward the ascorbate site.5 Support for an inhibitory binding of the monoanionic form of fumarate at theascorbate reductant site comes from velocity curves determined at low ascorbate (0.1 mM, Fig. 5) and high ascorbate (15 mM (36)) as afunction of variable fumarate. Although fumarate has historically been employed as an optimal activator for dopamine P-monooxygenase, this represents the first characterization of the dependence of its activation upon ascorbate concentration at variable pH values. As seen in Fig. 5, only at pH 6.7 does fumarate exert an activating effect from 0.1-10mM. At lower pH values, the inhibitory effects of fumarate become apparent at much lower concentrations (-0.2-0.4 mM), with the maximally activating level of fumarate observed to be -100-200 p ~The . presence of a much lower concentration of fumarate (100 p M ) at pH 5.2 does, in fact, lead to activation across the entire range of ascorbate concentrations (Fig. 6). Thebasic patterns observed with variable fumarate concentration using 0.1 M ascorbate and soluble enzyme (Figs. 5 and 6) have also been observed at high (15 mM) ascorbate and with the purified membranederived form of DPM (36). In the former case (15 mM ascorbate), the extent of inhibition is much reduced, as would be expected for a competitive effect of fumarate at theascorbate reductant site. The observation that only low levels of fumarate (100-200 p ~ are ) necessary to obtain activation of dopamine p-monooxygenase activity over a broad range of ascorbate concentrations isrelevant to virtually all previously published studies ofDPM kinetics in which high concentrations of fumarate (typically 10 mM) have been employed. Moreover, the apparently diminished curvature in kinetic plots at pH 6.0 in the presence of 10 mM fumarate may explain the results of Fitzpatrick et al. (22) in which a single K,,, for ascorbate was extracted from plots of velocities determined using 13 PM to 10 mM ascorbate. The very early data of Goldstein et al. (421, in which velocities were determined at pH6.0 in the presence of 10 mM fumarate, were in fact suggestive of the type of nonlinear behavior reported herein; however, ascorbate con-centrations greater than 2 mM were not examined. DISCUSSION
I
0
0
10
20
30
40
50
60
v/Iascorbate]
FIG. 4. Dependence of cooperativity upon oxygen concentration. Velocities were measured in the absence of fumarate from 0.1 to 20 mM ascorbate (H, 50% oxygen) and 0.2-20 mM ascorbate (+, 20% oxygen) in the presence of 10 mM tyramine hydrochloride.
The enzyme concentration was 5 pg/ml.
The datapresented in Figs. 1-4 provide strong evidence for cooperative behavior of ascorbate toward dopamine P-monooxygenase. The independence of the observed nonlinearity of Eadie-Hofstee plots with regard to substrate tyramine and oxygen levels (Fig. 4 and discussion under “Results”) appears to rule out a random binding phenomenon as the source of this cooperativity. Furthermore, the independence of cooperativity from changes in protein concentration (Fig. 3) indicates that these effects are unrelated to a dimer-tetramer The concentration of dissolved O2 in these experiments is 0.21 mM. Ascorbate, with pK, values of 4.5 and 11.8 (411, is expected to exist as a monoanion over the entire pH range.
Ascorbate Regulation
of Dopamine p-Monooxygenase
11541
20
16
12
8
.
04
.
, 10
0
.
.
.
, 20
.
.
I
30
.
I
4
40
vl(Fumnte1
0
40
0
20
60
80
100
vqascorbate]
FIG. 6. Effect of fumarate concentration upon variable ascorbate kinetic profiles at pH 5.2. Velocities were determined from 0.05 to 20 mM ascorbate in the absence (U) or presence of 100 p M (+) or 10 mM (0) fumarate (see Fig. 1 for comparison).
20
10
0
30
40
50
vpumarete] 3.0
c:
PH 6.7
Pathway 2:
[ASCI>
1 mM
SCHEME I. Random pathways for enzyme reduction by ascorbate, involvingfree enzyme at low ascorbate (Pathway 1) and the enzyme product complex at high ascorbate (Pathway 2).
1 .o 0
5
10
1s
20
a singleexponential process with a K,, of 0.9 mM for ascorbate FIG. 5. Effect of varying fumarate concentration on dopa- (26). Integration of the current body of knowledge concerning mine hydroxylase activity at low (0.1 mM) ascorbate. A fuDPM catalysis (cf. Refs. 9-11) with the currentfindings leads marate concentration range of 0.1-20 mM was examined at a tyramine for ascorbate activation; hydrochloride concentration of 15 mM and the pH values of 5.2 ( A ) , to theproposal of two possible models 5.5 ( B ) ,and 6.7 ( C ) .The control velocities determined in the absence these involve either a binding of ascorbate to an allosteric of fumarate were 6.2 (pH 5.2), 3.2 (pH 5.5), and 1.67 (pH 6.7) pmol/ regulatory site (Model 1) or the existence of multiple steady min .mg. Soluble enzyme was used at a concentration of 5 pg/ml. state forms of DPM which are capable of interaction with ascorbate as reductant (Model 2). An experimentalresult interconversion of DPM subunits. Although the behavior of relevant to Model 2 is the demonstration (by freeze quench fumarate is consistent with competition toward ascorbateat kinetics) that high levels of ascorbate reduce the enzyme its reductant site (Figs. 1 and 6), the observation that high product complex(39). Thisfindingcontrastswithearlier fumarate concentration still produces activation at saturating models for DPM, which had invoked an interaction of ascorascorbate (pH 6.7, Fig. 1B)indicates that the ascorbate reg- bate exclusively with the free enzyme form (11).As point out ulatory site is distinct from that of fumarate activation. in the studyof Brenner and Klinman (39), dual pathways are Previous results from this laboratory have led to a working likely for the reduction of DPM by ascorbate (illustrated as model forDPM catalysis involving a reductant copper binding pathways 1 and 2 in Scheme I). The kinetic importance of site which is separate from a second copper site catalyzing each of thesepathwaysis expected to reflect the rate of substrate hydroxylation (8, 26, 39). In this context, the sim- product release from E::$]. P, relative to rates for ascorbate suggest that low concentraplest explanation for the present findings would be an inter- binding and copper reduction. We action of ascorbate with both reductant and substratecopper tions of ascorbate may lead to a rate limitation by product sites, with these sites being characterized by vastly differing release from oxidized enzyme, such that measured K,,, and affinities and limiting VmaX values. This appearsvery unlikely, V,,,, values pertain to the interaction of ascorbate with free however, since it would require that reduction of copper at enzyme, pathway 1. Large elevations in ascorbate concentracompetencefor the weaker ascorbate binding site (presumably the substrate tionareexpectedtoincreasethekinetic site) give rise to a to 3-fold increase in activity. In any case, pathway 2. Thus, the high K,, region seen in Figs. 1-4 may previous freeze quench EPR studies of DPM with ascorbate represent the interactionof ascorbate with the enzyme prodshow rapid reduction of both enzyme-bound copper atoms in uct complex. The observed increase in turnover number can VIIFumarateJ
11542
Ascorbate Regulation
of Dopamine P-Monooxygenase
be rationalized from the chemistry of the rate-limiting step, which involves dissociation of product (as an alkoxide) from an inner spherecomplex with copper(52). It is tobe expected that reduction of copper from Cu(I1) to Cu(1) would weaken the binding interaction with product, leading to measurable increases in enzyme turnover. As an alternative to Scheme I, it is possible that DPM contains an allosteric regulatory site for ascorbate (Model 1). Weattemptedtotestthishypothesisthroughthe use of ascorbate 2-sulfate as a nonoxidizable analog of ascorbate. Although ascorbate analogs modified at the 2-hydroxyl position have been reported to retainreducing capability (53),we find no turnoverof DPM using ascorbate 2-sulfate as a putative reductant. Under conditions of 1mM ascorbate, inclusion of 30 mM ascorbate 2-sulfate in reaction mixtures has been found to producea rate increase of -1.7-fold; however, a similar rate increase was seen with potassium sulfate alone. This failure of a redox-inactive analog to ascorbate produce to a significant rate increase above control levels suggests an importance of redox chemistry in the observed activation by high ascorbate levels, Model 2. However, it is also possible that electrostatic and steric interactionspreclude an interaction of ascorbate 2-sulfate at a structurally restrictive allosteric site. Future reactivity studies, using a range of ascorbate analogs, may provide the information needed to distinguish unambiguously between Models 1 and 2. In an attempt to describe mathematically the dataof Fig. 1 minus fumarate, we used the model of Alberty et al. (23), which is based on a study of non-Michaelis-Menton kinetic behavior in the fumarase-catalyzed hydration of fumarate. As discussed (23, 54), a cooperative kinetic situation resulting either from substrate binding at a second site or from the existence of two different independent enzyme forms can be described by an equation comprised of four Michaelis constants, Vl and Kl (at low substrate concentration) andV, and K, (at high substrate concentration), to characterize thetwo kineticphases of theEadie-Hofstee(or double-reciprocal) plot.
TABLE I Final refined values for limited Vmx and K, parameters at low and high ascorbate concentrations forsoluble DpH Kinetic datafrom Fig. 1 (minus fumarate) and Ref. 36 were plotted in double-reciprocal fashion, and initial estimates of VI, Kl and V2, K2 were taken from the slopes and intercepts of the straight line portions of these curves a t low and high ascorbate concentrations. These estimates were refined by nonlinear least squares fitting of experimental data to Equation 2, using the algorithm of Marquardt, as described under “Experimental Procedures.” Vl and K1 represent limiting parameters a t low ascorbate concentration; Vz and Kz are limiting parameters a t high ascorbate. X* values reflect the sum of the squares of the deviations of the experimental data from the theoretical curve. The closer X’is to zero, the better thefit. pH
VI
Klb
V*-
mM
K2
X’
mM
0.073 42.12 5.2 12.69 23.5 5.5 9.82 0.049 32.15 16.3 5.95 8.42 0.088 34.90 48.2 6.70 2.73 0.051 11.78 36.3 a VI and V, have units of micromoles/min. mg. Kl and K2 have units of millimolar.
0.044 0.0025 0.0055 0.0043
TABLEI1 Final refined values for limiting Vmx and K,,, parameters at high and low ascorbate concentrations for purified membranous DOH Velocities were determinedas a function of variable ascorbate concentration(minusfumarate)using purified membranous DPH (36). Initial estimates of VI, K,, Vz, and Kz were obtained and refined as described in Table I for soluble enzyme data. pH
VI
KI
VZa
K*b
5.2 0.080 14.11 5.48 10.4 5.5 4.78 0.096 16.19 35.3 5.95 0.069 12.90 3.04 37.2 6.70 0.072 4.07 1.68 15.1 Vl and V, have units of micromoles/min. mg. Kl and K2 have units of millimolar.
X2
0.029 0.052 0.0036 0.0059
previous reports of a K, of 0.6 mM for soluble DPM (21, 22) and for DPM in chromaffin granules(50). The observationof a single, relatively low K , in these former studies is most likely a consequence of the rangeof ascorbate concentrations examined, thep H of the assay conditions and the presence of fumarate (cf. Fig. 6). Mennitiet al. (20) have already reported Using this equation, initial estimates of Vl, K1, V2, and K 2 a very highK, for ascorbate(15-17 mM) in intactchromaffin obtained for soluble DPM (Fig. 1 and Ref. 36) and membra- cells and granules, aswell as in granuleghosts. Findings from nous DBM (36) were refined using a nonlinear least squares our own laboratory confirm a high internal K,,, for ascorbate fitting routine togive the theoretical curves shown in Fig. 1. in chromaffin ghostvesicles (18). Final fitted parameters are shown in TablesI and 11. Whereas the data presented herein demonstrate a signifiTwo results emerge from these data. First,a comparison of cant activationof DPM in the rangeof intragranular ascorbic the limiting V,,, parameters at high and low ascorbate indi- acid concentrations (10-20 mM), the relevance of this phecates that, independent of pH, roughly 30% of V,,, for do- nomenon to enzymebehavior in vivo is unclear. There is pamine P-monooxygenase can be reached at low ( 4 mM) increasing evidence that the membrane-bound and soluble ascorbate concentrations. Second, the calculated K, values forms of DPM may undergodifferent mechanismsof electron for the portionof the kinetic curves representing high ascor- reduction (16-18). From the studiesof Huyghe and Klinman bate concentration are extremely large. At the intragranular (18),itcanbe concluded thatmembranousdopamine Pp H of 5.5 for chromaffin granules (43-45), K,,, = 35 mM for monooxygenase in chromaffin granule ghosts undergoes prefmembrane-derived DPM and 16 mM for the soluble enzyme. erential reduction by external ascorbate ( K , = 1.2 mM). In In the context of Model 1, this would support an allosteric the presence of internal ascorbate, the observed K,,, is much ascorbate site of substantially lower affinity than the ascor- higher (-29 mM), and themaximal rates of turnover are three bate reductant site. Alternatively (Model 2, Scheme I), these to four times less than those obtained with saturating external results imply much tighter bindingof ascorbate toE&‘@]than ascorbate (18).Furthermore, when both internal and saturatEg$ii.P; it is conceivable that the ascorbate and substrate ing external ascorbate are present, there is an activation of binding sites lie in sufficient proximity that steric hindrance approximately %fold over the rate observed with external impedes reductant binding in the presence of product. As ascorbate alone(18).This observationof a -3-fold activation of pointed out earlier (cf. Ref. 11), the absence of spin coupling by internal ascorbate is consistent with the present finding purified membranous between the reductant an$ substrate binding copper sites a 2.6- to 4.2-fold rate enhancement with indicates that theselie 2 4 A of one another. DPM (VI versus V,, Table 11) and suggests that in chromaffin We note that the K,,, values in Table I are in contrast to granuleghostsinternalascorbateinteractsprimarily with
Ascorbate P-Monooxygenase Regulation of DIopamine
11543
J. Biol. Chem. 260, 15598-15603 membrane-bound enzyme by binding at a n ascorbate regula14. Beers, M. F., Johnson, R. G., and Scarpa,A. (1986) J. Biol. Chem. tory site (cf. Ref. 18). 261,2529-2535 One striking feature that emerges from these studies is the 15. Menniti, F. S.,Knoth, J., and Diliberto, E. J., Jr. (1986) J. Biol. marked dependence of the apparent K,,, for ascorbate on the Chem. 261, 16901-16908 pHreaction 16. Ahn, N. G., and Klinman, J. P. (1987) J. Biol. Chem. 262,1485presence orabsence of anion activator and the of 1492 solutions (Figs. 1and 6). This propertyof ascorbate activation indicates that perturbations in reaction conditions will lead 17. Ahn, N. G., and Klinman,J. P. (1989) J . Biol. Chem. 264,1225912265 t o markedly different turnover ratesfor DPM. Ahn and KlinB. G., and Klinman, J. P. (1991) J. Biol. Chem. 266, man (18)have shown that the activityof DPM in chromaffin 18. Huyghe, 11544-11550 granule ghosts is very low (-10-fold lower than lysed mem- 19. Grouselle, M., and Phillips, J. H. (1982) Biochem. J. 202, 759brane sheets; -20-fold lower than detergent solubilized en770 zyme) and have suggested that the topographical constraints 20. Menniti, F. S.,Knoth, J., Peterson,D. S., and Diliberto, E. J., Jr. (1987) J . Biol. Chem. 262, 7651-7657 of forming a closed vesicle may render a large proportion of the membraneous enzyme unavailable for catalysis. Several 21. Kaufman, S.,and Friedman, S.(1965) Pharmacol. Reu. 17,71100 other workers (13, 19, 46, 47) have also observed low activity 22. Fitzpatrick, P. F., Harpel, M. R., and Villafranca, J . J . (1986) in both intact isolated granules and chromaffin cells in culArch. Biochem. Biophys. 249, 70-75 ture, suggesting that DPM may be a rate-limiting enzyme in 23. Alberty, R. A,,Massey, V., Frieden, C., and Fuhlbrigge, A. R. catecholamine biosynthesis. In this context, ascorbate acti(1954) J. A m . Chem. SOC.76, 2485-2493 vation of DPM could play a role in granule maturation. As 24. Klinman, J . P., and Krueger, M. (1982) Biochemistry 21, 67-75 noted, mature chromaffin granules contain extremely high 25. Stewart, L. C.. and Klinman. J. P. (1988) J. Biol. Chem. 263, 12183-12186 concentrations (-0.5 M ) of product norepinephrine (5,12). In 26. Brenner. M.C.. Murrav. C. J.. and Klinman. J. P. (1989) . , Bioorder to accumulate a store of product catecholamines as ’ chemistry 28,’ 46564664 rapidly as possible, the catalytic activityof DPM would need 27. Taljanidisz, J., Stewart, L. C., and Klinman, J. P. (1989) Biochemistry 28, 10054-10061 t o be maintained at ahigh level; ascorbateactivation of enzyme may be one possible mechanism of achieving such 28. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 29. Klinman, J . P., Krueger, M., Brenner, M., and Edmondson, D. high activity. E. (1984) J . Biol. Chem. 259, 3399-3402 There may be an equally important down-regulatory role 30. Bevington, P. R. (1969) Data Reduction and Error Analysis for for ascorbate following fusion of mature granules with the the Physical Sciences, pp. 235-236, McGraw-Hill, New York chromaffin cell membrane and release of intragranular small 31. Frieden, C. (1979) Annu. Reu. Biochem. 48,471-489 Methods Enzvmol. 64. molecules and soluble enzymes. As described (3, 4, 48), the 32. Neet. K. E.. and Ainslie. G. R... Jr. (1980) . 192-226 granule membranes (containing membranous DPM) are rapI. H. (1975) Enzymic Kinetics, pp. 67-71, John Wiley & idly retrieved from the cell surface and recycled to the Golgi 33. Segel, Sons, New York t o be repackaged with secretory proteins. During this retrieval34. Saxena, A,, and Fleming, P. J. (1983) J. Biol. Chem. 258,4147and transit process, it may be advantageous to maintain the 4152 activity of membranous enzyme at a low level. Although the 35. Colombo, G., Papadopoulos, N. J., Ash, D. E., and Villafranca, J. J . (1987) Arch. Biochem. Biophys. 252, 71-80 presence of -2 mM cytosolic ascorbate (49)is likely to lead t o some catalysis, the lack of intravesicular soluble enzyme and 36. Stewart, L. C. (1989) Structural and Kinetic Studies of Bouine Dopamine /3-Hydroxyluse. Doctoral thesis, University of Caliascorbate would serve to preventa high level of turnover. fornia, Berkeley Acknowledgment-We wish to thank Dr. Vern Schramm at the Albert Einstein College of Medicine of Yeshiva University for valuable discussions regarding possible kinetic origins of allostery. REFERENCES 1. Winkler, H., and Carmichael, S. W . (1982) in The Secretory Granule (Poisner, A.M., and Trifar6, J . M., eds) pp. 3-79, Elsevier Biomedical Press, Amsterdam 2. Winkler, H., Apps, D. K., and Fischer-Colbrie, R. (1986) Neuroscience 18, 261-290 3. Winkler,H., Sietzen, M., and Schober, M. (1987) Ann. N. Y. Acad. Sci. 493,3-19 4. Phillips, J. H., and Pryde, J. G. (1987) A n n N. Y. Acad. Sci. 493, 27-42 5. Carty, S. E., Johnson, R. G., andScarpa, A. (1985)in The Enzymes of Biological Membranes (Martonosi, A., ed) Vol. 2, pp. 449-495, Plenum Press, New York 6. Terland, O., and Flatmark, T. (1975) FEBS Lett. 59, 52-56 7. Ingebretsen, 0. C., Terland, O., and Flatmark, T. (1980) Biochim. Biophys. Acta 628, 182-189 8. Stewart, L. C., and Klinman, J. P. (1987) Biochemistry 26,53025309 T.(1984) in Copper Proteins andCopper 9. Ljones, T., and Skotland, Enzymes (Lontie, R., ed) Vol. 11, pp. 131-157, CRC Press, Boca Raton, FL 10. Skotland, T., and Ljones, T. (1979) Inorg. Perspect. Biol. Med. 2, 151-180 11. Stewart, L. C., and Klinman, J . P. (1988) Annu. Reu. Biochem. 57,551-592 12. Winkler, H. (1976) Neuroscience 1,65-80 13. Levine, M., Morita, K., Heldman, E., and Pollard, H. B. (1985)
37. Saxena, A., Hensley, P., Osborne, J. C., Jr., and Fleming, P. J. (1985) J . Biol. Chem. 260, 3386-3392 38. Dhawan, S., Hensley, P., Osborne, J. C., Jr., and Fleming, P. J. (1986) J. Biol. Chem. 261, 7680-7684 39. Brenner, M. C., andKlinman, J. P. (1989) Biochemistry 28, 4664-4670 40. Tinoco, I., Jr., Sauer, K., and Wang, J. C. (1985) Physical Chemistry: Principles and Applications in Biological Sciences, Prentice-Hall, Englewood Cliffs, NJ 41. Andrews, G. C., and Crawford, T . (1982) in Ascorbic Acid: Chem(Seib, P. A., and Tolbert, B. M., istry, Metabolism and Uses eds) American Chemical Society, Washington D. C. 42. Goldstein, M., Joh, T. H., and Garvey, T. Q., I11 (1968) Biochemistry 7, 2724-2730 43. Johnson, R. G., and Scarpa, A. (1976) J. Biol. Chem. 251, 21892191 44. Salama, G., Johnson, R. G., and Scarpa,A. (1980) J. Gen. Physiol. 75,109-140 45. Rottenberg, H. (1979) Methods Enzymol. 55, 547-569 46. Levine, M. (1986) J. Biol. Chem. 261, 7347-7356 47. Corcoran, J. J., Wilson, S. P., and Kirschner, N. (1984) J . Biol. Chem. 259,6208-6214 48. Patzak, A., and Winkler, H. (1986) J. Cell Biol. 102, 510-515 49. Levine, M., and Pollard, H. B. (1983) FEBS Lett. 158, 134-138 50. Dhariwal, K. R., Washko, P., Hartzell, W . O., and Levine, M. (1989) J . Biol. Chem. 264,15404-15409 51. Ahn, N., and Klinman, J. P. (1983) Biochemistry 22, 3096-3106 52. Miller, S.M., and Klinman, J. P. (1985) Biochemistry 24, 21142127 53. Kato, K., Terao, S.,Shimamoto, N., and Hirata, M. (1988) J . Med. Chem. 31, 793-798 54. Kistiakowsky, G . B., and Rosenberg, A. J. (1952) J . A m . Chem. SOC.74, 5020-5025
