I956

The pH-Dependence of Enzymic Ester Hydrolysis

By FELIX BERGMANN, RUTH SEGAL, ARELA SHIMONI AND MENACHEM WVURZELDepartment of Pharmacology, The Hebrew Univer8ity-Hadassah Medical School, Jerusalem, Israel

(Received 19 December 1955)

The esterases of animal tissues have generally beencharacterized by their substrate specificity and bytheir behaviour towards various inhibitors. Theirreversible inhibition by organic phosphates,which is due to phosphorylation of the active group(Burgen, 1949; Wilson & Bergmann, 1950a;Aldridge, 1953d), is a common property of mostesterases, an interesting exception being the A-esterase of Aldridge (1953a, b). This generalbehaviour suggests a similar structure of most ofthe esteratic groups responsible for the hydrolyticreaction.

True cholinesterase has been characterized, inaddition to the properties mentioned, by the dis-sociation constants of its esteratic site (Wilson &Bergmann, 1950 b). However, the negative chargespresent in the active centre of this enzyme arealso pH-dependent and thus interpretation of theexperimental data obtained with acetylcholine(ACh) as substrate is difficult (Bergmann & Shiimoni,1952 a). This difficulty can be met by studying theinfluence of pH changes on the enzymic hydrolysisof uncharged esters, since in such a system allvariations of activity can be ascribed solely tostructural changes of the esteratic site.

In the present investigation, such procedureshave been applied to true and pseudocholinesteraseand to an unspecific liver esterase. From theseexperiments certain conclusions about the chemicalstructure of the esteratic sites can be drawn, and arational interpretation can be derived for the bell-shaped curves relating hydrolytic rates to substrateconcentration which have been found not only in thesystem true cholinesterase-acetylcholine, but withother esterases and a variety of uncharged sub-strates (Bergmann & Shimoni, 1953). In addition,the dissociation constants of the anionic sites inboth types of cholinesterases have been determinedand a tentative explanation of the curious valuesobtained has been advanced.

Part of the results described in this paper havebeen communicated at the Meeting of the FaradaySociety held at Oxford in August 1955 (Bergmann,1955).

MATERIALS AND METHODS

Substrates. The esters used were commercial samples,purified by distillation. The preparation and properties ofthe fluoroacetates have been described previously (E. D.

Bergmann & Blank, 1953; Bergmann & Shimoni, 1953). Forthe pH range 5-8-5 the buffer used was 0-1 M phosphate, forhigher pH values 0-1 M borate. Sodium was the only cationpresent.Enzyme preparations. Two samples of true cholinesterase

were used. (a) A preparation from the electric organ ofTorpedo marmorata was obtained through the courtesy ofProfessor D. Bovet, Istituto Superiore de Sanita, Rome. Thepurified solution of the enzyme, when diluted 1:30 000,hydrolysed 9-6 .umoles/ml./hr., with 4 x 10-3M ACh assubstrate. (b) The enzyme from Electrophorus electricu wasa gift of Professor C. Chagaz, Department of Physiology,University of Brazil, Rio de Janeiro. The eel enzyme,diluted 1:1000, hydrolysed 6,umoles of ACh/ml./hr., withthe same substrate concentration as before.The pseudocholinesterase used was the crystalline

fraction IV-6-III from human serum, prepared in thelaboratory of the late Professor E. Cohn, HarvardMedical School, Boston. This preparation (0.1 mg./ml.)hydrolysed 8Mmoles/ml./hr., with 6 x 10-2M ACh assubstrate.

Dog-liver esterase. This was prepared as described pre-viously (Bergmann & Shimoni, 1952b). However, thecolour of the preparation interfered with the analyticalprocedure and its high protein content caused turbidity andprecipitation at acid pH. Therefore, the protein fraction,obtained from dog liver between 17 and 25% (w/v)(NH4)2S04, was dissolved in distilled water, brought to 15%(w/v) (NH4)2S04 and the precipitate discarded. The nextfraction, precipitating between 15 and25% (w/v) (NH4)2SO4,represented approximately two-thirds of the total enzymicactivity. It was further purified by repetition of the aboveprocedure, but fractionating within narrower limits, i.e.(a) 0-15%, (b) 15-20%, (c) 20-23% and (d) 23-25% (w/v)(NH4)2S04. The last precipitate (IlId), with a proteincontent of 16-6 mg./ml., was practically colourless andserved for the preparation of the stock solution. Whendiluted 1:100, it produced 6 6,4moles of C02/ml./hr., with0-43M diacetin as substrate.

It is noteworthy that the ratio of activity for diacetin andisopropyl fluoroacetate remained approximately constantduring the various purification steps, whereas the enzymichydrolysis of acetyl- and propionyl-choline decreasedsteadily. The liver enzyme IIId, which was used in thecolorimetric experiments, showed such a low activitytowards choline esters that measurements with these sub-strates had to be extended over long periods. Therefore,extrapolation of the enzymic activity to zero time, anecessary procedure in view of the competition between H+ion and cationic substrate (Wilson & Bergmann, 1950b),became unreliable.

Hydrolysis. This was measured by the method of Hestrin(1949), which was found applicable to alkyl halogenoacetateswith the modifications described in Table 1.

684

pH-DEPENDENCE OF ESTER HYDROLYSISEater concentrations. These were selected according to the

following considerations: (1) Initial solutions had to bemade without ethanol, which inhibits the enzymes studiedto various degrees (Bergmann & Shimoni, 1953). (2)Addition of the ester to the incubated mixture of enzymeplus buffer at zero time should not dilute this mixtureappreciably, in order not to disturb the equilibrium estab-lished between enzyme and hydrogen ions. (3) The finalconcentrations of substrate should be near that yieldingmaximal rates as determined previously (Bergmann &Shimoni, 1953). (4) The samples taken out for colorimetricdetermination should be large enough for accurate analysis,the upper limit being set by a colorimetric reading of 300 ona Klett-Summerson photoelectric colorimeter. (5) Theequilibrium between H+ ions and enzyme is disturbed bythe substrate, which competes for the active surface. There-

fore, the rate of hydrolysis has to be measured under non-

competitive conditions, i.e. by extrapolation to zero time.This requires sampling at short intervals. Thus, at pH 6-5-8samples were usually taken out at 1, 2, 3 and 4 min. In theacid range, increasingly longer reaction times had to beused, in view of the rapidly decreasing activity of theenzymes. In the alkaline range, such an adjustment wasnot feasible because of the spontaneous hydrolysis of theesters. The conditions used in these experiments are shownin Table 2.The rates were calculated by plotting ester hydrolysis as

a function of time and extrapolating to zero time. Thespontaneous hydrolysis was measured at the same time andsubtracted from the enzymic rates. The correction wasnegligible for n-propyl acetate at any pH. In the alkalinerange (above pH 9) all halogenoacetates underwent non-

Table 1. Colorimetric determination of ester8 as hydroxamic acids

The sample taken was made up to 1 ml., then 2 ml. of a mixture of equal parts of 2M hydroxylamine sulphate and3 5N-NaOH was added and the solution incubated, as indicated. Then 1 ml. each of 3-5N-HCI and of 0-74M-FeCl3, dis-solved in 0.1N-HCI, were added. Readings were taken immediately with a Klett-Summerson photoelectric colorimeter.Results are given per ,umole of ester/ml. of the final mixture.

Reaction with NH20H

Estern-Propyl fluoroacetateisoPropyl fluoroacetaten-Butyl fluoroacetateEthyl chloroacetaten-Propyl chloroacetateEthyl bromoacetaten-Propyl acetateAcetylcholine

Temp.(0)3035352727272727

Time(min.)10403044421

Klett units/j.mole/ml.

5958586060588298

Klett filterno.

5050505050505454

Fading rateof ferric

complex, Klettunits/min.

22310650

1

Table 2. Conditions of enzymic hydrolysis of various esters

Initial substrate concentration defines the standard solution prepared, portions of which were added to the previouslyincubated mixture of enzyme and buffer, so as to obtain the final substrate concentration. Enzyme dilution refers to thestock solutions, described in Methods.

Ester(1) Liver esterase

n-Propyl acetateisoPropyl fluoroacetateEthyl chloroacetate

(2) Serum cholinesteraseAcetylcholinen-Propyl fluoroacetaten-Propyl chloroacetate

(3) Torpedo esteraseAcetylcholinen-Propyl fluoroacetaten-Propyl chloroacetate

(4) Eel esteraseAcetylcholinen-Butyl fluoroacetateEthyl chloroacetateEthyl bromoacetate

Temp. Enzyme(0) dilution

24 1:3025 1:2724 1:50

36-5 1:300036-5 1:100037 1:1000

23 1:1000023 1:100023 1:500

24 1:12523 1:30023 1:3023 1:50

Substrate conen. (M)

Initial Final

3 x 10-21-5 x 10-17-5 x 10-2

10-19 x 10-22 x 10-2

6-6 x 10-29 x 10-22-4 x 10-2

5 x 10-23 x 10-27-5 x 10-29 x 10-2

1 x 10-22-5 x 10-22-5 x 10-2

10-23-6 x 10-210-2

3.3 x 10-33-6 x 10-28 x 10-3

5 x 10-310-22-5 x 10-22-4 x 10-2

Enzymichydrolysisat pH 7(,umoles/ml./min.)

Size ofsample(ml.)

1.0 0-31-4 0-21-2 0-2

0*5 0*30-75 0-150-6 0.5

0*5 0.80-4 0.150-6 0-6

0-8 0-60-6 0.52-0 0-22-2 0-2

Vol. 63 685

F. BERGMANN, R. SEGAL, A. SHIMONI AND M. WURZELenzymic hydrolysis, the descending order being bromo-,chloro-, fluoro-acetate. All figures were expressed as per-centage of the maximum rate of the enzyme-substratesystem under consideration.

RESULTS

Dia8ociation con8tants of the eBteratiC 8ite8

In Fig. 1 the results with dog-liver esterase havebeen summarized. All curves are similar and demon-strate that a polar substituent in the acyl portionof the ester has little influence on the position of thepH optimum, which for this enzyme is rathersharply defined. Acidic and basic dissociationconstants of the esteratic site are thus practicallyindependent of the type of substrate used. Bothisopropyl fluoroacetate and ethyl chloroacetatehave been found previously to possess a pS ( - log S,where S is the substrate concentration) maximum(Bergmann & Shimoni, 1953), showing that in the

system liver esterase-halogenoacetate a non-hydrolysable ES2 complex is formed by excess ofsubstrate.We can thus describe the system by the same four

equilibria, applied previously to the combinationtrue cholinesterase-acetylcholine (Wilson & Berg-mann, 1950b), and use the equations derived therefor the determination ofpKa and pKb, the dissocia-tion constants of the nucleophilic and the electro-philic groups of the esteratic site. The figuresobtained in this way are represented in Table 3.When applying the same procedure to pseudo-

cholinesterase, we have to take into account thepresence of an anionic site (Bergmann & Wurzel,1954; Bergmann & Segal, 1954), which becomesprogressively inactivated below pH 7 by recombi-nation with H+ ions. However, with unchargedsubstrates we may neglect as a first approximationthe influence of the anionic site on ester hydrolysisat the esteratic site or the changing polar forces

100

-80, -

.60

40 Ar/

-Z20

5 6 7 8 9pH

Fig. 1. pH-dependence of enzymic hydrolysis by dog-liveresterase. 0, Ethyl chloroacetate; x, isoPropyl fluoro-acetate; 0, n-Propyl acetate. Enzyme and substrateconcentrations are given in Table 2. Buffer used forpH <8-5 is 0-1 M phosphate, for pH >8-5, 0-1 M borate, astheir sodium salts.

100

~80 ~

ei i/6/

20 /

v40 ~ ~ \

H 91

Fig. 2. pH-dependence of enzymic hydrolysis by human-serum cholinestera-se. 0, Acetylcholine; 0, n-propylchloroacetate; x, n-propyl fluoroacetate. For details ofexperimental conditions see Table 2.

Table 3. Dissociation constants of the components of the active centre of various esterases

EnzymeDog-liver esterase

Human-serum cholinesterase

True cholinesterase fromTorpedo marmorata

True cholinesterase fromElectrophoru electricus

Substrate or inhibitorEthyl chloroacetateisoPropyl fluoroacetaten-Propyl chloroacetateTetraethylammonium bromiden-Propyl fluoroacetateTetraethylammonium bromideEthyl chloroacetateEthyl bromoacetateTetraethylammonium bromide

pK. pKbof esteratic site6-1 9.46-8 9-2

pK ofanionic site

6-2 9-0- - 6-56-2 9-0

6-36-7 9-66-5 9-0

- 6-2

686 I956

pH-DEPENDENCE OF ESTER HYDROLYSISbetween the anionic site and the halogen atom. Weapply therefore the same equations as above.Indeed, justification for this procedure can be foundin the similarity of the pH activity curves in Fig. 2with those in Fig. 1. The dissociation constants,derived for human-serum cholinesterase, are in thesame range as those for liver esterase (see Table 3).However, the curve for ACh deviates from thosefor all other substrates, mainly in the alkalinerange. This phenomenon, which is probably relatedto the interaction of the cationic group in ACh withthe anionic site, is discussed below.

S-1X0,o0

Elkg

0

50c

EEx

:

/1.|

6 7pH 85 9

Fig. 5. pH-dependence of the inhibitory effect of tetra-ethylammonium bromide on cholinesterases. *, Eelesterase 1:125, ACh 4 x 10-3m, Et4N+ 5 x 10-'M;0, Torpedo esterase 1: 5000, ACh 4 x 10-3M, Et4N' 2-5 x10-2 M; x, serum cholinesterase (quoted from Bergmann& Wurzel, 1954).

80

0X10 60

El~40

13 20

5 6 7 pH 8 9

Fig. 3. pH-dependence of enzymic hydrolysis by Torpedoesterase. x, Acetylcholine; 0, n-propyl chloroacetate;0, n-propyl fluoroacetate. For details of experimentalconditions see Table 2.

The two representatives of true cholinesterase,used in our experiments, show a different pHdependence (Figs. 3, 4). The Torpedo enzyme issimilar to serum cholinesterase, but for the systemeel esterase-n-butyl fluoroacetate a shift of the pHoptimum to about 9 has been observed. Thisbehaviour is shared by the ethyl and n-propyl estersand may be ascribed to an interaction of thefluorine atom with a group at the enzyme surface.

Di88ociation con8tant8 of the anionic 8ites

For the evaluation of these constants the pH-dependence of inhibition by tetraethylammoniumbromide has been used. The enzyme and a constantconcentration of inhibitor were incubated withbuffers of various pH for 1 hr., then ACh was addedand hydrolysis determined after 1, 2, 3 and 4 mi.The rates obtained by extrapolation to zero timewere compared with the inhibitor-free controls forthe calculation of the percentage inhibition. Themaximum inhibitory effect (between pH 7 and 9)was then put equal to 100 and the results in the acidpH range were expressed as a percentage of thismaximum, to compare different sets of experimentswith different enzymes (see Fig. 5). The descendingbranches ofthe curves in Fig. 5 have been treated astitration curves to evaluate the apparent dissocia-tion constants of the anionic sites. The figuresderived in this way are also included in Table 3.

DISCUSSION

5 6 7 8pH

Fig. 4. pH-dependence of enzymic hesterase. 0, Acetylcholine; x, ethylethyl bromoacetate; A, n-butyl fi

details of experimental conditions see

The results in Table 3 demonstrate that for allesterases used in the present study the dissociation

9 10 constants of the esteratic site, pK, and pKb, fallwithin a rather narrow range. This supports the

ydrolysis by eel assumption that the active group, responsible forchloroacetate; , hydrolysis, is essentially the same in all of them.

Luoroacetate. For Since no prosthetic group has been found inTable 2. esterases, we may assume that the components of

Vol. 63 687

F. BERGMANN, R. SEGAL, A. SHIMONI AND M. WURZEL

the active centre are amino acids. On this basis, thevalue ofpK. - 6 5 suggests the presence of histidine,the iminazole group of which is in equilibrium withits cation and possesses a pK of 6-7 in variouspeptide combinations (Cohn & Edsall, 1943). Itshould be noted that our value for pK. is lower thanthe corresponding value of pKHt+ = 7 2, derivedpreviously from the system eel esterase-ACh(Wilson & Bergmann, 1950b).The second dissociation constant, pKb ';9 3, has

been interpreted previously to indicate an a-aminogroup (Bergmann, 1955). However, in peptides thepK of this group acquires a value of about 7 1-8-2(Ellenbogen, 1952). Our previous assumption thusneeds correction. It now appears likely that thegroup responsible for the second dissociation con-stant is represented either by the p-hydroxyl groupof tyrosine (pK in various peptides, 9.3-9.8) or, lessprobably, by the E-amino group of lysine (pK,9.6-10.7). In the former, we would have to assumethat the un-ionized phenolic group is the active formand that dissociation into the phenoxide ion destroysthe enzyme activity. For the e-amino group oflysine, on the other hand, we would have to concludethat only the ammonium form is active and that itsconversion into the free base abolishes enzymicactivity.

If, as is usually assumed, the active portion of theenzyme molecule is composed of amino acids only,the anionic sites are most probably represented bythe free carboxyl group of aspartic or glutamic acid(Bergmann, 1955). However, the ,B-carboxyl of theformer in various peptides has a pK of 3 0-4-5, andthe y-carboxyl of the latter shows a pK of 4-2-4-5.Thus, the experimental values found (see Table 3)are 2-3 units too high, but are surprisingly near thepK value of the iminazole group. This suggests thatwe are measuring, in our experiments concernedwith the pH variation of the anionic site (Fig. 5),not its inactivation by recombination with HI ions,but its association with the iminazolinium ion oftheesteratic site. The anionic site is, so to speak,titrated by iminazolinium ion. In the true enzyme,we would have to assume that both carboxylategroups combine electrostatically with the samecation. The probability of the formation of suchion complexes is enormously increased on thesurface of a protein molecule, because both anionand cation have only restricted freedom of motion,and the water, bound to the surface, behaves likea medium of low dielectric constant. In thisconnexion it is of interest that neutralization of thetwo anionic sites in the true enzyme occurs at asomewhat lower pH than that of the single negativecharge in the pseudo enzyme. In the former, theiminazolinium ion has to compete with two positiveions in the surrounding solution.To summarize, we conclude that the active site of

esterases comprises at least two amino acid residues,namely histidine and tyrosine. For pseudocholin-esterase one residue of glutamic (or aspartic) acidhas to be included, for the true enzyme two. In thelatter, the two glutamate fragments may either beseparated by one or more of the other amino acidsrequired or they may be adjacent to each other.A model based on the latter alternative has beenconstructed and shows that in this case the distancebetween the two negative charges is of the appro-priate size to accommodate such a bis-quaternaryion as decamethonium.The structure of the active surface, suggested by

the above considerations, sheds light on severalexperimental facts, which have so far eluded arational explanation.

(1) A bell-shaped pS-activity curve is observednot only for the system true cholinesterase-ACh,but also for the other esterases used in the presentexperiments, especially when halogenoacetatesform the substrates (Bergmann & Shimoni, 1953).It should be noted that in these cases a second polarsubstituent is usually present in the molecule inaddition to the ester grouping. Apparently, thisfavours the formation of an ES2 complex. If thereare two polar groups available, which can combinewith corresponding polar portions of the proteinmolecule in the neighbourhood ofthe active surface,the affinity ofthe ester molecule becomes sufficientlylarge to replace water on the active surface. (Withinthe active centre, we assume, a small number ofwater molecules required for hydrolytic reactionsare being held tightly.) Accordingly, when theaffinity of a given ester type is increased bylengthening the carbon chain of the alcoholicportion, the pS optimum shifts to higher values(Bergmann & Shimoni, 1953). It is of great interestthat pseudocholinesterase cannot form an ES2compound with ACh, but does so with a sub-strate like ethyl fluoroacetate. This finding againstresses the importance of the second anionicsite in the true enzyme, which provides theanchor to which the second molecule of ACh canbe attached.

(2) A further problem arises from consideration ofthe relative rates of hydrolysis within the homo-logous series of choline esters. With the pseudoenzyme, the rate increa-ses with increasing length ofthe acyl side chain, the values reported being acetyl-choline :propionylcholine: butyrylcholine = 1: 1 5: 2(Augustinsson, 1949). On the other hand, for thetrue enzyme the relative rates are approximately1: 0 9:0 01, the absolute values differing somewhatfor different preparations (e.g. Aldridge, 1953c;Nachmansohn & Rothenberg, 1945). Butyryl-choline (BuCh) is, however, well adsorbed by theactive surface of the true enzyme, as indicated bythe concentration (2.5 x 10-3M) required to produce

688 I956

pH-DEPENDENCE OF ESTER HYDROLYSIS

50% inhibition of ACh hydrolysis (Bergmann &Shimoni, 1953). This shows that it is the step in-volving reaction of BuCh with water that does notproceed at a satisfactory rate. We may express thisfact by saying that for true cholinesterase auto-inhibition of hydrolysis occurs with ACh, when twomolecules combine with the active surface, whereaswith BuCh one molecule produces the same effect.We shall therefore try to explain these two pheno-mena on a common basis. From a model of theactive surface built from the four units mentionedabove, it became apparent that the space availablearound the esteratic site is very restricted, beinglimited by the presence ofthe two anionic groups, sothat the amount of water that can be adsorbedsimultaneously with a substrate molecule may be-come the critical factor. Therefore, with a short acylchain as in ACh, the second substrate moleculecompetes with the water necessary for reaction.With BuCh, the acyl chain of one ester mole-cule is already long enough to replace practi-cally all the water which can be adsorbed onthe active surface and thus prevents its ownhydrolysis.The curves for ACh in Figs. 2-4 show rather

broad pH maxima. This is especially pronouncedwith eel esterase, in accordance with the observa-tions of earlier investigators (Hestrin, 1950; Wilson& Bergmann, 1950b). In the pH range of 5-7 thecurve for ACh deviates little from that for thehalogenoacetates examined, but a pronounceddifference appears on the alkaline side, where thedecay of enzymic hydrolysis is delayed for ACh.This is another expression of the fact that theanionic sites play a major role in the combinationenzyme-ACh. As has been calculated previously(Bergmann & Segal, 1954), the coulombic forcesrepresent about 90% of the total binding force ofACh to true cholinesterase. Therefore the affinity ofthe cationic substrate for the anionic sites outweighs,to a certain extent, the decrease in affinity producedby inactivation of the electrophilic group in theesteratic site. In the acid pH range such an effectcannot be observed, owing to the phenomenonmentioned above, namely, the iminazole group,becoming inactivated by combination with H+ion, neutralizes the anionic sites.

These assumptions are supported by the followingobservations: we have tried to construct the pH-activity curve of ACh in Figs. 2 and 4 by super-imposing the dissociation curve of the anionic sitesas exemplified in Fig. 5, and the pH-activity curveof an uncharged ester, namely ethyl chloroacetate.Although the selection of just this substrate isarbitrary, similar if somewhat less satisfactoryresults are obtained with the other halogenoacetates.In Fig. 6 the superimposed curves are compared withthe experimental data.

44

A surprising shift of the pH optimum has beenobserved for the system eel esterase-alkyl fluoro-acetate (see Fig. 4). Since the correspondingphenomenon is absent with the Torpedo enzyme(and all other esterases), this may indicate thatnear the active centre of the eel esterase a group ispresent with a high affinity for fluorine, probably byhydrogen bonding, which thus outweighs to acertain extent the decrease in affinity occurring inthe alkaline pH range.As a final point, we should mention that in

degradation studies of various hydrolytic enzymes,including cholinesterases, after their irreversibleinhibition by dii8opropyl phosphorofluoridate

100t (a)

.-o

80

- 60c400

tv40,4,

@ 20ce

lk

lF

I I I I I9 105 6 7 pH8

5 6 7 8 9 10 11pH

Fig. 6. Comparison of the experimental data for the pH-dependence of the system cholinesterase-ACh withtheoretical values. The theoretical points represent thearithmetic mean of the values in Fig. 5 on one hand andthe values for chloroacetate in Fig. 2 (serum cholinester-ase) or Fig. 4 (eel esterase) on the other. (a) Plasmacholinesterase. 0, Experimental; *, calculated. (b) Eelesterase. 0, Experimental; x, calculated.

Bioch. 1956, 63

Vol. 63 689

690 F. BERGMANN, R. SEGAL, A. SHIMONI AND M. WURZEL I956(DFP), Schaffer, May & Summerson (1953, 1954)and Oosterbaan, Kunst & Cohen (1955) found theorganic phosphate linked to a serine residue. It hasbeen shown by Jandorf, Michel, Schaffer, Egan &Summerson (1955) and by Hobbiger (1955) that theinhibitory reactions with phosphates pass throughtwo reaction steps, so that one may assume that thephosphate-serine linkage represents the final stageof the interaction between enzyme and inhibitor.It is difficult to see how the hydroxyl group ofserineparticipates in ester hydrolysis, since a spon-taneously reversible acylation of the esteratic siteis required (Bergmann, Wilson & Nachmansohn,1950). In addition, the dissociation constants oftheesteratic site give no information about the possibleparticipation of serine. Nevertheless, one has toadmit that the active centre of an enzyme maycomprise units which do not undergo any changewithin the pH range measurable for the enzymicreaction. Therefore the problem of a possible role ofserine in enzymic ester fission remains unsolved atpresent.

SUMMARY

1. The change of hydrolytic rate with pH hasbeen studied for different types of esterases andwith a variety of uncharged (non-cationic) sub-strates. From these measurements, the dissociationconstants of the nucleophilic (pKI) and the electro-philic (pKb) group of the esteratic site have beencalculated for each enzyme-substrate system.

2. The esteratic sites of all esterases studiedpossess approximately the same dissociation con-stants. The chemical structure ofthe active surfacesof various esterases is thus closely related.

3. The dissociation constants of the negativesites of true cholinesterase and pseudocholin-esterase have been derived from the pH-dependenceof the inhibitory effect of tetraethylammonium ion.The curious values obtained, 6-2-6-5, seem toindicate neutralization at acid pH of the anionicsites by the iminazolinium ion of the esteraticgroup.

4. Possible structures of the esteratic sites havebeen discussed, and explanations put forward forthe inhibitionbylarge substrate concentration ofthehydrolysis of cationic and uncharged esters and forthe different behaviour of true cholinesterase andpseudocholinesterase towards the homologous seriesof aliphatic choline esters.

REFERENCES

Aldridge, W. N. (1953a). Biochem. J. 53, 110.Aldridge, W. N. (1953b). Biochem. J. 53, 117.Aldridge, W. N. (1953c). Biochem. J. 53, 62.Aldridge, W. N. (1953d). Biochem. J. 54,442.Augustinsson, K. B. (1949). Arch. Biochem. 23, 111.Bergmann, E. D. & Blank, I. (1953). J. chem. Soc. p. 3786.Bergmann, F. (1955). Di8c. Faraday Soc. 20, 126.Bergmann, F. & Segal, R. (1954). Biochem. J. 58, 692.Bergmann, F. & Shimoni, E. (1952a). Biochim. biophys.

Acta, 9, 473.Bergmann, F. & Shimoni, E. (1952b). Biochim. biophys.

Acta, 8, 520.Bergmann, F. & Shimoni, E. (1953). Biochem. J. 55, 50.Bergmann, F., Wilson, I. B. & Nachmansohn, D. (1950).

J. biol. Chem. 186, 693.Bergmann, F. & Wurzel, M. (1954). Biochim. biophy8.

Acta, 13, 251.Burgen, A. S. V. (1949). Brit. J. Pharmacol. 4, 219.Cohn, E. J. & Edsall, J. T. (1943). Proteins, Amino Acidsand Peptides. New York: Reinhold Publ. Corporation.

Ellenbogen, E. (1952). J. Amer. chem. Soc. 74, 5198.Hestrin, S. (1949). J. biol. Chem. 180, 249.Hestrin, S. (1950). Biochim. biophys. Acta, 4, 310.Hobbiger, F. (1955). Brit. J. Pharmacol. 10, 356.Jandorf, B. J., Michel, H. 0., Schaffer, N. K., Egan, R. &Summerson, W. H. (1955). Disc. Faraday Soc. 20, 134.

Nachmansohn, D. & Rothenberg, M. A. (1945). J. biol.Chem. 158, 653.

Oosterbaan, R. A., Kunst, E. D. & Cohen, J. A. (1955).Biochim. biophys. Acta, 16, 299.

Schaffer, N. K., May, S. C. jun. & Summerson, W. H. (1953).J. biol. Chem. 202, 69.

Schaffer, N. K., May, S. C. jun. & Summerson, W. H. (1954).J. biol. Chem. 206, 201.

Wilson, I. B. & Bergmann,F. (1950a). J. biol. Chem.185,479.Wilson, I. B. &Bergmann,F. (1950b). J.biol. Chem.186, 683.