Proc. Nati. Acad. Sci. USAVol. 80, pp. 2107-2111, April 1983Biochemistry

Chemical evidence that catecholamines are transported across thechromaffin granule membrane as noncationic species

(catecholamine transport/dimethylepinephrine/isoproterenol/norepinephrine)

AVNER RAMU*, MARK LEVINEt, AND HARVEY POLLARDt*Depament of Clinical Oncology, Hadassah Medical School, Jerusalem, Israel; and tSection on Cell Biology and Biochemistry, Laboratory of Cell Biology andGenetics, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205

Communicated by Elizabeth F. Neufeld, December 22, 1982

ABSTRACT Catecholamines are transported into chromaffingranules by a Mg2+/ATP-driven process under conditions in whichthe substrate exists primarily as a positively charged or neutralspecies. In order to distinguish between these two states, we stud-ied the transport properties of a permanently charged quaternaryanalogue of epinephrine, (R,S)-dimethylepinephrine. We foundthat this compound was a classical competitive inhibitor of (R)-[3H]norepinephrine uptake, with a K; of 3.8 mM for the racemicform, or 1.9 mM for the R form. However, the [3H]dimethyl-epinephrine was not transported at all into granules. Our controlfor steric hindrance as an explanation for deficient translocationwas analysis of the transport properties of isoproterenol, a sec-ondary catecholamine with an isopropyl group around the amineresidue. (R)-Isoproterenol was an effective competitive inhibitorof (R)-[3H]norepinephrine transport, with a K; of 91 ,uM. In con-trast to dimethylepinephrine, (RS)-[3H]isoproterenol was clearlytranslocated across the granule membrane, with a Km of 123 aiM,or 61.5 IAM for the R isomer. Thus, the positive charge on di-methylepinephrine and not the size of the amine moiety appearedto be responsible for the lack of translocation. We interpret thesedata to indicate that, although the positively charged species caninteract with the transport site, an uncharged species is the oneactually transported.

Catecholamines are transported into chromaffin granules by aMg2+/ATP-driven process, which has become increasingly at-tractive as a general and simple system to analyze chemiosmoticenergy coupling to transport (1-5). Many models have beenproposed to explain the process, but distinguishing among themhas been made difficult by lack of knowledge of whether theneutral, cationic, or anionic catecholamine species is the truesubstrate (4, 6-9). Recently, on the basis of observations thatboth the binding constants for dopamine and serotonin and thecalculated cationic fractions were constant between pH 6.8 and7.6, Knoth et al. (10) concluded that the cationic species wastranslocated. However, using a similar experimental approach,Scherman and Henry (11) came to the opposite conclusion. Theyobserved that the Km for norepinephrine declined between pH6.5 and 8.5, and they interpreted this as due to an increase inthe concentration of the uncharged species, the proper sub-strate. Yet, on the basis of entirely different considerations,Johnson and Scarpa (7) initially claimed that the species trans-ported was most likely neutral, and more recently summarizedby saying that a conclusion was not yet possible (8).We therefore decided to reinvestigate this question directly

by examining the transport properties of a quaternary analogueof epinephrine. The compound we used was (R,S)-dimethyl-epinephrine (Me2E). * Me2E and its tritium-labeled derivativehave the structure shown in Fig. 1. Because of its quaternary

AOH H CH3

f-N IHO H'- C-N-CH3

IHO H H CH3

BOH H H CH3

HO C- -H H C-H

)/ I Io1+HO H H H CH

FIG. 1. Structures of Me2E (A) and isoproterenol (B). [3H]Me2E waslabeled at the p carbon (*). The wavy line on isoproterenol inm denotesthe bond broken when the first proton dissociates. For norepinephrine(which has no methyl groups on the nitrogen atom) the pK is 8.57, andfor epinephrine (one methyl group) the pK is 8.73 (12, 13).

structure, this compound exists only as the positively chargedspecies.

Our intention in these experiments was to see if Me2E couldfunction as a competitive inhibitor of norepinephrine transport,and if so whether Me2E could itself be translocated into gran-ules. If the cationic species were transported as proposed byKnoth et al. (10), then competitive inhibition and transport couldbe observed. As a control for the size of the substituted aminogroup on Me2E, we also studied the closely related structuralanalogue isoproterenol (Fig. 1). Isoproterenol is also a true cate-cholamine (12); in it the secondary amine has a three-carbon-atom residue attached to the amine moiety, and it is isomericto Me2E. Isoproterenol thus exists as a mixture of cationic, an-ionic, neutral, and zwitterionic species, similar to conventionalphysiologic substrates. Johnson et al. (14) have also recentlyshown isoproterenol to be taken up by chromaffin granules inan ATP-activated, reserpine-sensitive manner, just like naturalsubstrates. However, the detailed kinetic properties of isopro-terenol have not been extensively evaluated (4, 14-16).

In the present study we report that Me2E is indeed a classicalcompetitive inhibitor of (R)-norepinephrine transport. How-ever, we also found that the inhibition constant was extremelyhigh and that [3H]Me2E was not transported. By contrast, iso-proterenol was both a competitive inhibitor and a substrate fortransport. We conclude that the cationic species is not trans-ported and indeed is not even preferred over uncharged cate-cholamine species in terms of affinity for the transport site.

MATERIALS AND METHODSPreparation of Chromaffin Granules. Chromaffin granules

were prepared by differential centrifugation in 0.3 M sucroseas described in earlier studies from this laboratory (17, 18). Thepurified granules were maintained on ice at a concentrationequivalent to an optical density of 5.0 at 540 nm for no morethan 15 min prior to the initiation of the experiment.

Abbreviation: Me2E, dimethylepinephrine.t Dimethylepinephrine and its 3H derivative were used as the R,S (ra-cemic) mixtures, as was [3H]isoproterenol; isoproterenol (unlabeled)and norepinephrine (unlabeled and 3H-labeled) were of the R config-uration.

2107

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Proc. Natl. Acad. Sci. USA 80 (1983)

Assay of Transport into Chromaffin Granules. Transportassays were performed exactly as described in our recent paper(19). Briefly, granules in a volume of 0.25 ml were added to3.25 ml of prewarmed reaction mixture and incubated for dif-ferent times. The reaction mixture contained 50 mM Hepes/NaOH buffer at pH 7.5, 1 mM MgCl2, 1 mM Na2ATP, 20 mMKC1, different amounts of unlabeled catecholamines, and enoughsucrose and water to bring the system to 335 milliosmolar. Aconstant amount of radioactivity, 1 ,uCi (1 Ci = 3.7 X 1010 Bq)per tube (total volume of 3.5 ml at initiation of the experi-ments), was used in each appropriate reaction mixture, exceptfor experiments concerning uptake of [3H]Me2E. For uptake of[3H]Me2E, the reaction mixtures contained 10 tkCi per tube.The reaction was terminated by addition of 2 ml of ice-cold0.335 M sucrose. Subsequent manipulations were done at 40C.Granules were pelleted at 32,000 rpm in a Sorvall SS2Y rotorfor 20 min and washed one or more times in 0.335 M sucrosebefore further analysis. One milliliter of de-ionized water wasadded to each pellet. Pellets were frozen and thawed with agi-tation prior to determinations of radioactivity, protein, andcatecholamine.

Synthesis and Analysis of Me2E. Me2E iodide [(R,S)-di-methylaminomethyl(3,4-dihydroxyphenyl)carbinol methio-dide] and its precursors were prepared for us by Regis Chem-ical (Chicago) (contract no. 278-0035) under the direction of Al-bert Manion (National Institute of Mental Health), accordingto a three-step method (20). First, a-dimethylamino-3',4'-di-hydroxyacetophenone hydrochloride was prepared (mp 206-208°C). Then a Parr shaker was charged with 14.0 g (0.06 mol)of a-dimethylamino-3',4'-dihydroxyacetophenone hydrochlo-ride, 400 ml of ethanol, and 0.80 g of platinum oxide. The sus-pension was shaken under 50 pounds/inch2 (340 kPa) of hy-drogen for 4 hr; the catalyst was removed by filtration; and thefiltrate was concentrated to dryness, treated with 10 ml of con-centrated ammonium hydroxide, and extracted with 125 ml ofethyl acetate six times. Concentration to 25 ml and filtrationyielded 5.70 g (48.3%) of dimethylaminomethyl(3,4-dihydroxy-phenyl)carbinol (mp 142-1430C). Finally, Me2E was preparedby addition of 3 ml of methyl iodide to a solution of 0.40 g (0.002mol) of(R, S)-dimethylaminomethyl(3,4-dihydroxyphenyl)carbi-nol in 15 ml of ethanol. The solution was stirred 1 hr and al-lowed to stand at room temperature for 16 hr, and diethyl ether(15 ml) was added to the cloud point. After 10 min, the solidwas filtered and dried to yield 0.437 g (63.5%) of Me2E iodide(mp 180-1820C). Elemental analysis of Me2E iodide corre-sponded to the calculated formula of C11H181NO3. The productwas homogeneous by thin-layer chromatography performed onWhatman silica gel with 1-butanol/acetic acid/water, 50:25:25(vol/vol). The NMR spectrum measured in H20 with a 100-MHz JEOL FX100 spectrometer was consistent, with regard toposition, coupling, and integrated intensities, with the pro-posed structure.

Synthesis and Analysis of [3H]Me2E. [3H]Me2E was pre-pared by catalytic reduction of a-dimethylamino-3,4-dihy-droxyacetophenone methachloride with tritium gas by UriBuchman of Nuclear Research Centre, Negev, Israel. Purifi-cation was accomplished by extraction with ether/water. At thetime of synthesis chemical purity was tested by analytical thin-layer chromatography and by UV spectrophotometry. The sol-vent systems were the one described above and methanol/l-butanol/benzene/water, 40:30:30: 10 (vol/vol). [3H]Me2E was98% pure by these two analyses. Thin-layer chromatographicanalysis of [3H]Me2E was identical to that of Me2E as describedabove. Immediately prior to experimentation, both Me2E and[3H]Me2E were found by high-performance liquid chromatog-raphy (21) to behave as a single homogeneous peak. The spe-

cific activity of [3H]Me2E was 4.27 Ci/mmol.Chemicals and Assays. Protein was determined by the Brad-

ford assay (22), as described for application to chromaffin tissue.Catecholamines were analyzed by the spectrofluorometricmethod of Anton and Sayre (23). Chemicals were of reagentgrade and were used without modification. Other isotopicallylabeled compounds were purchased from New England Nu-clear and consisted of (R)-[3H]norepinephrine (30.6 Ci/mmol)and (R,S)-[3H]isoproterenol (1.6 Ci/mmol).

RESULTSInfluence of Me2E on (R)-[3H]Norepinephrine Transport.

Transport of (R)-[3H]norepinephrine proceeds linearly for morethan 20 min over a concentration range of 14-228 tLM (R)-nor-epinephrine (19). Using this system, we found that Me2E in-hibited (R)-[3H]norepinephrine uptake in a concentration-de-pendent manner. In the present paper kinetic analysis over a20-min period with different concentrations of Me2E revealedthat only the apparent rate of (R)-[3H]norepinephrine uptakewas reduced. For 80 different experimental conditions the av-erage r value for the linear uptake curves was 0.991 ± 0.008.The nature of the inhibition by Me2E could then be analyzed

by a Dixon plot as shown in Fig. 2. We observed that the dif-ferent substrate concentration curves intersected in the upperleft quadrant, coincident with the perpendicular projection ofthe 1/Vmn, point. This indicated that the inhibition was com-petitive, and that the Ki for Me2E, estimated from the projec-tion to the x axis of the intersection point, was 3.8 mM. If theinhibition were due exclusively to the R form of Me2E, as forother catecholamines (19), then the Ki would be 1.9 mM. Weinterpreted these data to indicate that, although affinity of Me2Efor the transport site was low, Me2E nonetheless was capableof interacting with this site.

Transport Properties of [3H]M2E. Having shown that Me2Eis recognized as a competitor at the transport site, we wonderedwhether the compound itself was capable of being transported.[3H]Me2E was prepared and shown to be identical to unlabeledMe2E under a variety of separation procedures as outlined inMaterials and Methods. However, we found that under no con-dition could we induce chromaffin granules to internalize thecompound. Representative transport data under a variety ofconditions are shown in Fig. 3 Upper, with an example of (R)-

1 2 3 4Me2E, mM

FIG. 2. Dixon plot of inhibition of (R)-[3H]norepinephrine uptakeby Me2E. (R)-[3H]Norepinephrine concentrations were 28.6 jLM (curveA) and 114.3 MtM (curve B). Uptake velocity is given in units of nmol/mg of protein per min.

2108 Biochemistry: Ramu et al.

Proc. Natl. Acad. Sci. USA 80 (1983) 2109

0.06

0o0.4

C 0.02

= 40

30 -

0202

lo C00 5 10 20

Time, min

FIG. 3. (Upper) Uptake of [3H]Me2E into chromaffin granules.The concentration of [3H]Me2E was 0.67 pM. Conditions include noMgATP, 370C (curve A), 1 mM MgATP, 370C (curve B), 1 mM MgATP,00C (curve C). Addition of 3.6 mM MeWE or-285 PiM (R)-norepinephrineyielded curves parallel to and slightly below curve C (data not shown,for clarity). Error bars indicate SEM. (Lower) Experiment performedat the same time as that in Upper, showing that the preparation canaccumulate (R)-[3H]norepinephrine (28.6 u.M) (curve A, o) in a reser-pine-sensitive manner (curve C, A). In a separate experiment, inclusionof (R)-[3Hlnorepinephrine (28.6 PiM) and [3H]Me2E (0.67 /AM) in thesame reaction mixture demonstrated that (R)-[3H]norepinephrine up-take (curve B, e) proceeded normally.

[3H]norepinephrine transport included for comparison in Fig.3 Lower. Whereas about 40 nmol/mg of protein of (R)-nor-epinephrine can be accumulated in 20 min by chromaffin gran-ules, essentially none of the [3HJMe2E was transported overthe baseline of 0.035 nmol/mg of protein, with or withoutMgATP, and at 0C or 37C. In this attempt to observe uptakeof [3H]Me2E, we added 2.2 x 10 dpm (0.67 AM) of pure la-beled compound to the reaction mixture. If the Km were equalto the K, (3.8 mM) and if a Vmax equal to that for norepineph-rine was assumed, then we calculated that we should have ob-served an increment over the baseline of 225,000 dpm in a re-,action mixture containing 0.5 mg of protein. Thus, if transporthad occurred we could have measured it easily.When 3.6 mM unlabeled Me2E was added, no uptake of

[3H]Me2E greater than that at the initial time point was ob-served.

As an added control we added both 0.67 AM [3H]Me2E and28.6 ,uM [3H]norepinephrine, observing (Fig. 3 Lower) that la-beled Me2E did not interfere with uptake of the true substrate.We concluded that although Me2E would interact with thetransport site it was clearly not transported.

Transport Properties of Isoproterenol. .The key questionposed by the results with Me2E was whether the charge or sizeof the amino function was the main factor limiting transport.Isoproterenol seemed to be an ideal compound to distinguishbetween these two properties. It has three bulky methyl groupsnear the nitrogen atom as Me2E does, but has the advantageof being a secondary amine and therefore without a permanentpositive charge (Fig. 1). (R,S)-[3H]Isoproterenol was rapidly takenup by a process that was linear for 30 mm and saturable withrespect to (R, S)-isoproterenol concentration (see Fig. 4). The

0aa)S .-

0 1,

0t:6

Go.,.

as v

,s/

-

IS

cd

6

50 100 200 300

(R.S)-[3HlIsoproterenol, /iM

FIG. 4. Substrate-velocity plot of uptake of (R,S)-[3H]isoproter-enol into chromaffin granules. (Inset) Lineweaver-Burk plot of data inthe figure.

Km was 123 ,uM, a value only 2-3 times that of the Km we ob-served for (R)-norepinephrine. Because the isoproterenol iscomposed of a mixture of isomers and it is likely that only theR form is transported (19), then the true Km is probably 61.5AM. This calculated Km is nearly identical to the Km of 49-57AuM we observed for (R)-norepinephrine (data not shown) as wellas the Km of 49 AuM previously reported from this laboratory(19). In addition, our calculations based on the R form as thespecies preferentially transported show that the Vma, for (R)-iso-proterenol is 4.15 nmol/mg of protein, which is within a factorof 2 of the Vmax for (R)-norepinephrine (19).

0 57.14 114.29 228.58(R)-Isoproterenol, ,uM

FIG. 5. Dixon analysis of inhibition of (R)-[3H]norepinephrine up-take by (R)-isoproterenol in chromaffin granules. Curve A, 28.6 PM(R)-norepinephrine; curve B, 114.3 1utM (R)-norepinephrine; curve C,228.6 pM (R)-norepinephrine. Uptake velocity is given in units of nmol/mg of protein per min.

Biochemistry: Ramu et al.

r

Proc. Natl. Acad. Sci. USA 80 (1983)

As expected from its transport properties, (R)-isoproterenolalso inhibited the uptake of (R)-[3H]norepinephrine. The Dixonplot in Fig. 5 showed the inhibition to be typically competitive,with a Ki of 91 piM, substantially less than that for Me2E. Asan additional test for the possible inhibitory power of the cat-ionic quaternary amino group in Me2E we also tested cholinechloride (HOCH2CH2N+(CH3)3 Cl1-) Choline chloride did notin any way affect transport, at concentrations as high as 3.7 mM.Therefore, it seems apparent that the charge of the amino groupand not the size of Me2E is of fundamental importance in pre-venting Me2E from being a successful substrate for the chro-maffin granule transport site.

DISCUSSIONThe transport studies reported here show that Me2E, a per-manently cationic catecholamine, binds to the catecholaminetransport site on the chromaffin granule membrane but is nottranslocated across the membrane. Studies on the analogues(R)-norepinephrine and (R,S)-isoproterenol support the con-clusion that it is the charge property of the Me2E amino grouprather than its size that is responsible for the unusual behaviorof Me2E. As summarized in Table 1, (R)-norepinephrine and(R)-isoproterenol have KMi Vmax, and Ki values that are withina factor of 2 of one another. Because it is likely that only theR form of catecholamine is transported (19), then the two com-pounds seem to be nearly identical in terms of kinetic param-eters. The profoundly different properties of Me2E regardingcompetitive inhibition and transport compel us to conclude thatthe permanent cationic charge on the molecule is responsible.

Our conclusions can lead to a clearer understanding of theproblem of catecholamine transport mechanisms in the chro-maffin granule system. Transport is activated by the hydrolysisof ATP, which leads to the inward movement of a proton andthe generation of an inwardly positive electrical potential (6, 17,24-26). The basic problem remains, as in other chemiosmoticenergy-coupled transport systems, as to how the energy is cou-pled to transport (4, 25, 27). Of great importance to under-standing energy coupling is knowledge of the charge of thecatecholamine that is transported.

In fact, this particular aspect of chromaffin granule cate-cholamine transport, the nature of the transported species, hasoccupied many investigators (4, 6-10). Johnson and Scarpa (6,7) proposed that dopamine might be transported into granulesas the neutral species, analogous to ammonia equilibration acrossbiological membranes with subsequent -trapping by protona-tion. However, these experiments may not be relevant to ATP-driven catecholamine transport, which occurs at much lowercatecholamine concentrations. Later studies on the ability ofchromaffin granule ghosts to transport catecholamines by usingonly electrical potential AP in the absence of ApH argued against

Table 1. Comparison of transport kinetics of (R)-norepinephrine,(R)-Me2E, (R)-isoproterenol, and choline in chromaffin granules

V.axInhibition of nmol/mguptake of Ki, K., protein

Species (R)-norepinephrine ,uM ,uM per min(R)-Norepi-nephrine Competitive 49 49 6.8

(R)-Isopro-terenol Competitive 91 61.5* 4.15*

(R)-Me2E Competitive 1,890* None NoneCholine

chloride None None* Data have been corrected for the R isomer of catecholamine.

the ion trapping model (9). Indeed, Johnson and Scarpa con-cluded recently that the charge of the transported catechol-amine could not be determined from the information available(8).The idea of the neutral species being transported has also

been questioned by Njus and colleagues, who have suggestedthat the cationic species is the true substrate (10). Their con-clusions were based on two observations. First, the measuredKm values for uptake of serotonin and dopamine were constantover the pH range of 6.8-7.6. Second, the calculated mole frac-tions of serotonin and dopamine in cationic form were also con-stant over the same pH range. The interpretation of these ob-servations was that the cation was the true substrate for transport.In view of our data, it is also possible that variation in externalpH affected membrane functions, because the pH dependencefor transport titrates with a pKa of approximately 6.4 (28), whereasnorepinephrine titrates at higher pK. values (8.57, 9.73, 11.13)(13). In addition, the rate of interconversion of catecholaminespecies may be rapid compared to the rate of binding or trans-port. Indeed, our data show that binding and transport are notidentical, a distinction also alluded to by Johnson et al. (14).Thus, while differences in interpretation may remain, the ac-tual observations of Knoth et al. are not necessarily inconsistentwith our own. Indeed, Scherman and Henry (11), using anidentical experimental approach to that of Knoth et al., usedanother substrate over a wider pH range and came to conclu-sions similar to ours.Our work, directly demonstrating the cationic species to be

bound but not transported, can now be used to devise morerefined models of the transport process. Our model, like themodels of Njus and Radda (4) and Johnson and Scarpa (8), as-sume that the driving force for transport is the proton electro-chemical potential, ASH+ = At + FApH (F, Faraday con-stant). ASH+ is physiologically created by inward electrogenictransport of H' by the membrane ATPase. In addition we as-sume that the mechanism is electrically dissipative, althoughthe evidence for this is indirect. We use "electrically dissipa-tive" to mean that the net charge across the membrane is zeroafter one complete transport event. Furthermore, we take intoaccount that, in the intact granule, transport can occur eitherby net accumulation or by a 1:1 exchange process in which theuptake of an exogenous catecholamine is accompanied by therelease of an endogenous catecholamine (19). Finally, our trans-port model, as well as the models of others, postulate that trans-port occurs at a specific site and is carrier mediated.

As shown in Fig. 6, our model accounts for all of the con-ditions necessary for catecholamine transport. The first eventin line 1 is ATP-dependent translocation of a proton into thechromaffin granule, generating a charge of + 1 compared to thegranule exterior. In both lines 2A and 2B the catecholaminethat actually enters the chromaffin, granule is the neutral spe-cies. Line 2A shows the possibility that the charged species ini-tially binds to the membrane carrier, with subsequent disso-ciation of a proton prior to transport, while line 2B simply showsthe neutral species initially binding. At this point we cannotexclude either of these alternatives with the data at hand, butthe charge balance is still + 1 inside because the neutral cate-cholamine enters as the transported species. Lines 3A and 3Baccount for the dissipation of the positive charge by exchangemechanisms. Either a charged catecholamine may carry out aproton, as in line 3A, or there are separate exiting mechanismsfor the catecholamine and the proton, as in line 3B. Althoughwe have no data relating to the charge of the species leaving thegranule, both mechanisms account for proton efflux and res-toration of charge balance to 0. Line 4 illustrates a mechanismfor dissipation of the positive charge by release of a proton,

2110 Biochemistry: Ramu et al.

Proc. Natl. Acad. Sci. USA 80 (1983) 2111

FIG. 6. Electrodissipative models for catecholamine transport bychromaffin granules. Each line corresponds to an event in the model,beginning (line 1) with ATPase-driven H+ transport, which causes anincrement in internal charge of +1. Line 2 depicts alternative mech-anisms by which an uncharged catecholamine can penetrate the gran-ule membrane. Line 3 shows how charge can be dissipated, either byexchange or by a combination of exchange and H+ extrusion. Line 4illustrates how net accumulation of catecholamines and charge dissi-pation can be. accomplished by H+ extrusion. See text for more completedescription.

leaving the catecholamine inside. Such a mechanism would ac-

count for net accumulation.We believe that our model accurately describes granule

transport that is driven by a protonmotive force and that is elec-trically dissipative. The model accounts for net transport as wellas exchange. Thus the representations depict catecholaminetransport in terms of the fundamental assumptions we have out-lined. We anticipate that the use of a quaternary catecholamineto evaluate the character of the transported species may proveto be of general value.We are extremely grateful to Dr. Herman Yeh for NMR analysis of

Me2E and to Dr. David Goldstein for high-performance liquid chro-

matography analysis of Me2E and [3H]Me2E. We thank Bernard Lan-caster for his technical assistance and Elise Urciolo and Joan Mok forpreparation of the manuscript.

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Une Process Charge Balance

OUT IN

2A CatH+ - Cat H + tC0 CatH + 1

.~~~~ ~_ .1 _

B CatH -' Cat -' CatH + 1

H + lHH+I

3A Cat H + .- CatH + -- Cat H + 0

B CatH -Cat4 Cat CatH , l

4 H+ H+ 0

Biochemistry: Ramu et al.