Carboxylesterases (EC 3.1.1). A comparison of some kinetic properties of horse, sheep, chicken, pig, and ox liver carboxylesterases.

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Carboxylesterases (EC 3.1.1). A Comparison of Some Kinetic Properties of Horse, Sheep, Chicken, Pig, and Ox Liver Carboxylesterasesl Departmenr of Biarcite~nistry~ Uitiversity of Queet~slcii~d, St. L~tciu,Qiwenslcincl4067, Alrstrclliea Receivod January 28, 1974 Stoops, J. K., Hamilton. S. E. B Zerner, B. 61975) Carboxylesterases (EC 3.1.1 ). A Comparison of Some Kinetic Properties of Horse, Sheep, Chicken, Pig, and Ox Liver Carboxylesterases. Can. J . Biocitem. 53, 565-573 A comparative study of the kinetic behavior of horse, sheep, chicken, pig, and ox liver carboxylesterases is reported. The enzymes exhibit similar specificities towards a series of phenyl esters in which the acyl group is varied, and towards a series of butyrate esters in which the alcohol group is varied. Non-Michaelis-Menten kinetics are exhibited by the horse enzyme in the hydrolysis of methyl and ethyl butyrates, and by the pig enzyme with ethyl butyrate. Each enzyme exhibits inhibition by one or more substrates. A simpk scheme which accounts for both activation and inhibition is discussed. pH-kcat profiles for the horse and chicken liver carboxylesterase-catalyzed hydrolyses of phenyl butyrate demonstrate dependencies on pKals of 4.75 and 5.8, respectively. Stoops, J . K., Hamilton, S. E. & Zerner, B. (1975) Carboxylesterases (EC 3.1.1). A Comparison of Some Kinetic Properties of Horse, Sheep, Chicken, Pig, and Ox Liver Carboxylesterases. Can. J. Biochern. 53, 565-573 Nous avons fait une dtude comparative du cornportement cinttique des carboxylesterases hdpatiques du chevaI, du mouton, du poulet, du porc et du boeuf. Ces enzymes ont la meme spCcifacitC vis-i-vis divers phdnyl esters diffdrant par leur groupement acyle et divers butyrate esters diffdrant par leur fonction alcool. L9enzyme du cheval laisse voir des comgorternents cindtiques non Michadliens lors de l'hydrolyse du mdthyl- et de 19Cthylbutyrate. 11 en est de rn2me pour l'enzyme du porc avec l'dthyl butyrate. Chaque enzyme est inhibee par un ou plusieurs substrats. Nous discutons d'un schema simple qui tient compte et de l'activation et de l'inhibition. Les profils pH-kc,, des carboxylestCrases hdpatiques du cheval et du poulet lors de 19hydrolysedu phdnyl butyrate dCmontrent la ddpendance de ces enzymes h des pK,' respectifs de 4.75 et de 5.0. [Traduit par le journal]

Introduction The mammaIian liver carboxylesterases (EC 3.1.1.1) form a group of closely rklated enzymes, notably with respect to their molecular properties (f,2). The enzymes catalyze the hydrolysis of a wide variety of carboxylic acid esters and thislesters, and also of some aromatic amides (3). The best substrates appear to be simple aliphatic and aromatic esters of n-alkyl fatty acids. Because of the limited data available, a detailed comparison of the kinetics of the carboxylesterases is not possible. Further, any comparison must take into account the non-Michaelis\

'This work was supported in part by the Australian Research Grants Committee (Australia) and by grant GM 13759 from the Institute of General Medical Sciences of the U.S. National Institutes of Health. =A.R.G.C. Postdoctoral Fellow. Present address: Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77025, U.S.A. 3Commonwealth postgraduate student.

Menten behavior of some of the esterases. Substrate activation of pig liver carboxylesterase has been reported by several groups of workers (4-7). Similar behavior has been reported for horse liver carboxylesterase; Burch ($) obtained results for the hydrolysis of methyl butyrate which are consistent with substrate activation, and Hofstee (9) reported substrate activation with m-carboxyphenyl esters of fatty acids. This contrasts with a report by Adler and Kistiakowsky (4) that the horse enzyme exhibits MichaelisMenten kinetics in the hydrolysis of methyl butyrate. Inhibition of the carboxylesterases by substrate has also been reported. Murray (10) observed inhibition of sheep and rabbit liver carboxylesterases at high concentrations of ethyl butyrate. Stoops et al. (5) reported the inhibition of the pig and ox liver enzymes by phenyl acetate, and further investigation in this laboratory has revealed inhibition of the ox enzyme by ethyl


566

CAN. J. BIQCHEM.

butyrate (Inkerman, P. A. : unpublished observations). In this paper we report the results of iiavestigations of the kinetic properties of highly purified horse, sheep, and chicken liver enzymes; the results are compared with those obtained previously ( 5 ) for pig and ox liver carboxylesterases. Materials and Rlethods Ren,qefzts

VOL. 53,

1975

substrate concentration investigated in the absence of acetonitrile were: ethyl and methyl butyrates, 0.5-50 mM; nitrophenyl butyrates, 0.02-0.22 mM; phenyl acetate, 0.05-9 T M ; phenyl valerate, 0.05-5 mM: phenyl pivalate, 0.05-0.8 mM. The concentration of enzyme in each assay was -80-8-10-9 N. Molar absorption coeficients for the phenols were determined under the assay conditions. The maximum velocity, V, and the Michaelis constant, 6(,, were usually obtained by Lineweaver-Burk or Eadie analysis of the experimental data (initial rates) using the method of least squares. The precision of measured values of kc,, and K, (Tables 1 3 ) , indicated by the standard deviations in V and K,,, is k2('i and k5C/,. respectively, except when indicated otherwise. Data obtained for the hydrolysis of the L- and D-isomers of Z-tyrosine p-nitrophenyl ester4 were analyzed by the method of Menri (Dixon and Webb (86)).

Acetonitrile (Spectro grade) was obtained from the Eastman-Kodak Co., Rochester. N.Y. Paraoxon was purchased from Koch-Light Laboratories (England) and Tris (Trizma Base, reagent grade) from Sigma Chemical Co. All salts and buffers were analytical grade reagents. Buffers were filtered through a sintered-glass filter before use. Chicken, horse, and-sheep liver ia~box~lesterases were purified as described in the preceding papers (1 1,12). Results The horse and slaeep enzymes were stored as anamonium sulfate precipitates at pH 7.5. Samples were dialyzed Butyrate Esters against 0.01 M Tris buffer, 0.05 NaCl (pH 7.51, to Values of k,,t and K,, for the enzyme-catalyzed provide stock solutions for kinetic experiments. When hydrolysis of a series of butyrate esters in the the activity of a stock solution had decreased by 3';) it was discarded (generally in about 3 weeks). Chicken liver absence of acetonitrile are given in Table 1. The enzymes studied were horse, sheep, and chicken carboxylesterase was stored in 0.01 lV1 Tris butfer, 0.05 M NaCl (pH 7.5) for the duration sf the experianents, liver carboxylesterases. Kinetic constants for the without significant loss of activity. The i~ormalitiesof the 2,4-dinitrophenyl ester are also included, alenzyme solutions were determined by titration as previthough a minimum of 37, acetonitrile was reously described (1 3). The horse and sheep enzymes were titrated with 61-nitrophenyl dimethyl carbamate (12) and quired in these runs. Data obtained in the the chicken enzyme wa.; titrated with paraoxoil 611). presence of 12.8y0 acetonitrile for chicken liver These normalities were used in the calculation of kc,, arboxylesterase are given in Table 2, and comvalues. The specific activities of the enzymes used in this with values previously obtained for the ox, work were: chicken 4000, horse 2667, sheep 4500 (pkat! pig, and sheep enzymes. l),'A280. The equivalent weights of these enzymes measured during the kinetic runs were: chicken 68 800, horse Eflect q#'Acyl Group on Reactivity 69 800, sheep 69 500. All substrates were prepared as The enzyme-catalyzed rates of hydrolysis of a described previously (5, 14). Spectral analysis demonstrated that phenolic esters contained less than I,: of the series of phenyl esters were determined. The corresponding free phenol. enzymes used. in this study were the horse and Rule ~VIt~a.~uren~elats

Titrimetric assays were performed at 25.0 f 0.1 C by a pH-stat method using a Radiometer TTTlc, SBR2c, and SBUla (Copenhagen) (15). Rates were measured at pH 7.6 in the presence of 0.1 lV1 NaCI. Spectrophotometric assays were made using a Cary 14 recording spectrophotometer at 25 f 0.1 "C.An aliquot of the substrate in acetonitrile was added to 3 nal of buffer in a 8 c n ~cell. followed by an aliquot of the enzyme. The final concentration of acetonitrile in each assay, except when indicated otherwise, was 12.8:';. Alternatively, the assay was performed in the absence of acetonitrile, by addition of 250 of concentrated buffer to 3 ml of a water solution of the substrate. BufTers used in studying the pH dependence of kc,, and Knl for the enzyme-catalyzed hydrolysis of phenyl butyrate were: pH 3.5-6.1, acetate; 6.1-7.5. phosphate; 8.1-8.5, Tris; 9.6-18.5. carbonate. Soclium chloride was added to acetate and Tris buffers, so that at all pH's the final ionic strength was 0.1. The approximate ranges of

sheep liver carboxylesterases. Corresponding data for the ox and pig liver enzymes and achymotrypsin are also included (Table 3). Hydroly~isof D- and L-Esters Values of k,,t and K,, for the sheep and horse liver carboxylesterase catalyzed hydrolysis of the D- and L-isomers of N-benzyloxycarbonyltyrosine p-nitrophenyl ester are given in Table 3, together with the data for pig liver carboxylesterase and a-chymotrypsin.

pH-Rate Profiles The effect of pH on the horse and chicken liver carboxylesterase catalyzed hydrolysis of phenyl butyrate was studied in the absence of acetsni4Abbreviation used : 2. benzyloxycarbonyl.

567

STOOPS ET AL.: CARBOXYLESTEWASES. 1v

TABLE I . Hydrolysis s f butyric acid esters by horse, sheep, chicken, and pig liver carboxylesterases at 25 ' C

koa ( i V - I s-l)a

Horse

Sheep

Chicken

Pheny1 p-Nitrophenyl o-Nitrophenyl 2,4-Binitrophenyl Ethyl

0.69 5.9 2.8 23

204; 0.77h 144; 1 . l b 233; 0.50b 6 4 ; 0.298 135; 10.6h.j

565; 0.640 267; 0.430 366; 0.430 173; 0.100 559; 2.5h.j

476; 2.02cpd 51.7; 0.55cJ 245; 1 .41c.f

520; 2.7a~e

299;

7.7"

551; 7.2k 252; 8.35"

286 ; 58 . g h

454; 2.6"j

261;

18.P


Butyrate

Methyl

Pig

aStoops et al. 45). bQ.037 M phosphate (pH '7.62). c0.05 M phosphate @H 7.6). 2 4%. W;n f 7%. ?pH-stat measurement, p H 7.5. fkat 3 % , L k 8%. 00.036 iM phosphate (pH 7.67) and 3 % acetonitrile. hpH-stat measurement, pH 7.6 (0.1 M NaCI). jKm 10%. kpH-stat measurement, pH 7.5. Substrate activation obserked. hoe's anti Knm'sfrom high [S] data (1.6-23 m.M) and low [S] data ( 0 . 0 9 4 . 2 mM). NOTE;6, 6, g, are Cary 14 spectrophotorneter measurements.

+

+

TABLE2. Hydrolysis s f butyric acid esters in butfers containing 12.8'\;: acetonitrile, by carboxylesterases at 25 "C

kc,, (s-l); 1 0 T m (Mj Butyrate

Sheepuld

Chickene

Pigo -

Phenyl p-Nitropheny1 o-Nitrophenyl

105; 14 100; 20 $ 9 ; 17

226; 4.48 9 7 ; 3.7 226; 8 . 3 -

-

" ~ a ~ oXo.o.d t*) -

519; 20f 367; 14f 348; 17g

-

160; 17 110; 22 210; 6

-

aStoops et ul. (5). h6'Fast" specifies a single electrophoretic variant of the ox enzymc. c0.05 M phosphate (pH 7.6). d0.1 IW phosphate (pH 7.45). ekcat 9 3%,Kn, 6%. f0.05 M phosphate (pH 7.42). 80.15 M Tris (pH 8.1).

*

trile. Values of kcatand K, at each pH are given in Table 4. Each system is clearly dependent on a single pK,, and the pK,'s obtained from a graph sf log kcatvs. pH were 4.75 and 5.8 for the horse and chicken enzymes, respectively.

Substrare Inhibition A decrease in the catalytic rate constant at high substrate concentratione was observed for the following enzyme-substrate combinations, in the absence of acetonitrile: sheep liver carboxylester- pheny1, P - ~ ~ ~ ~ nitrophenyl butyrates, phenyl acetate, phenyl valerate; horse liver carboxylesterase - s-nitrophenyl butyrate, phenyl acetate; chicken liver carboxylesterase - methyl, ethyl butyrates. Kinetie Scheme 1 is consistent with all the substrate inhibition data summarized above.

-I-S -t-S E & E S e SES

In this scheme, the enzyme substrate complex ES decomposes with a rate constant k l and the complex SES is The ~ P ~ ~ equation ~ Y ~ steady-state for ,the scheme is given in Eq. 1.

k &b [E1O[sl K,Kb Kb[S] [SI2 At low substrate concentrations this simplifies to Eq. 2. [I]

=

+

+

(M3-1)

aFrom k o b , 0.05 M Tris (pH 7.0-9.0). b0.036 M phosphate (pH 7.62) and 1.53 % acetonitrile. ekeat f 3773, Km k 10%. d0.11 M phosphate (pH 6.59) and 0.8 % acetonitrilc. eStoops et al. (5). fDetermined in standard sodium hydroxide containing 1.2% acetonitrile. 00.037 M phosphate ( p H 7.62). h0.053 M Tris (pH 8.30), 0.18 M NaCI. j0.05 M phosphate (pH 7.42) and 12.8% acetonitrile. kQ.01 M phosphate (pH 7.45) and 12.8% acetonitrile. 10.037 M phosphate (pH 7.62) and 5 '3$ acetornitrile. W.1 M phosphate (pH 7.45) and 3% acetanitrile. W.09 M phosphate (pH 6.18) and 12.8% acetonitrile (14). 00.1 M phosphate (pH 6.96) and 3% acetonitrile (84). D0.034 M phosphate (pH 7.79) and 8.5% acetonitrile. N9.041 M phosphate (pH 7.2) and 8.6% acetonitrile. rQ.072 M phosphate (pH 7.68) and 8.5% acetonitrile. s0.072 M phosphate (pH 7.75) and 8.4% aectonitrile.

Phenyl formate Pkenyl acetate Phenyl butyrate Phenyl valerate Phenyl pivalate 2-Fkenyloxazolin-5-one Z-L-tysosinep-anitrophemyl ester ZD-tyrosinepnitrophenyl ester

Substrate

OH Horse

Sheep

Carboxylesterase

TABLE 3. IERect of acyl group on reactivity at 25 "C

Pig

Ox


a-Chymotryypsin

STOOPS EB AL. : CARBOXY LESTEWASES. BV

TABLE 4. pH dependence of kc,, and Km for carboxylesterase-catalyzed hydrolysis of phenyl butyratea Horse kpat


PH

Chicken

(s-'1;

lo4dg,(MI

pH

k c s t (§-I); 104 K , (MI

Hn this scheme, substrate binds first to a site other than the active site to form SE, and then to the active site, to form the activated complex SES, which decomposes with a rate constant k2? The steady-state equation for this scheme is Eq. 4.

At high substrate concentrations, Eq. st simplifies to the Miihaelis-Menten form (Eq. 5).

a25 "C. bAcetate buffer. CPhosphate buffer.

A Lineweaver-Burk plot of the data at Iow substrate concentrations gives ter and Ka. At high substrate concentrations Eq. f becomes:

k 1 and Kb may be evaluated from a graph of l l v vs. IS] using the data at high substrate concentrations. Some calculated values of KI, are given in Table 5. The corresponding values of 1%1, (= Km)and kl (= kcbat) are given in Tables f and 3. The values of Ka,KI,,and kl thus obtained were employed to calculate v at any substrate concentration using Eq. I. The predicted values of v were in close agreement with the experimental values at aU substrate concentrations, clearly supporting the proposed scheme. Substrate as Mod@er A rapid decrease in the zero-order rate was observed at low substrate concentrations in the horse liver esterase-catalyzed hydrolysis of me thy1 butyrate ([S]" < 3 m M ) and ethyl butyrate ([SIB < 1.5 a). Kinetic Scheme 2 is consistent with these results.

Hence k2 and Kd may be evaluated. Using these values and the experimental values of r9 at low substrate concentrations, K, may be calculated from Eq. 4 (Table 6). Discussion Specija'eity That the esterases, by analogy with the serine proteinases, catalyze the hydrolysis s f esters via an acyl-enzyme intermediate is an attractive hypothesis. While it has been possible to demonstrate the participation of an acyl-enzyme directly in some proteinase-catalyzed hydrolyses (17), the high reactivity and the instability of esterases at low pH have so far precluded such studies with the latter group of enzymes. A simple kinetic scheme which describes the acyl-enzyme hypothesis may be written

5An equally valid but no more illuminating description would be to say that the first molecule of substrate binds to the active site but is not hydrolyzed unless another molecule of substrate occupies a modifier site: adherence of initial rate data to such a kinetic scheme cannot possibly comment on a mandatory sequence of binding.

CAN. J. BIBCHEM. VOL. 53, 1975

TABLE5. Inhibition constants for horse, sheep, and chicken liver carboxylesterases


10qb (IM), carboxyiesterase Substrate

Horse

Sheep

Chicken

Phenyl butyrate p-Nitrophcnyl butyrate o-Nitrophenyl butyrate Ethyl butyrate lkiethyl butyrate Phcnyl acetate Phenyl valerate

No inhibition No inhibition -6.1 No inhibition No inhibition

3.7 -3.4 -22.8 7.3

No inhibition No inhibition No inhibition I3 -4

-

No inhibition

-42

0.23 -3.7

17.6 -

FABLE 6. Horse liver carboxylesterase-catalyze$ hydrolysis of methyl and ethyl butyrates, calculation of Kc [Substrate] ( m M ) Ethyl butyrate

Methyl butyrate

Indirect kinetic evidence which is consistent with the above scheme has been obtained for ox and pig liver carboxylesterases ( 5 , 18-28). The data of Table I indicate that if a butyryl-enzyme is formed as a common intermediate in the hydrolysis of this series of butyrate esters, its decon~positionis probably not the rate-limiting step in most cases. The equality of kcat's for the sheep and pig esterase-catalyzed hydrolysis of ethyl and phenyl butyrates is, however, not inconsistent with a (largely) rate-limiting deacylation for these substrates. Substitution of a nitro group on the aromatic ring of phenyl butyrate reduces k,,t and K,, in the order phenyl > o-nitrophenyl > p-nitrophenyl > 2,4-dinitrophenyl for all the esterases except for the horse enzyme, where kcat for s-nitrophenyl butyrate > kcat for phenyl butyrate. Malhotra and Philip (21) obtained similar results for the goat intestinal esterase-catalyzed hydrolysis of butyrate esters Qphenyl > p-nitrophenyl > s-nitrophenyl), and Stoops (22) observed that k,,t's for the acetylcholinesterasecatal-wed hydrolysis of a series of acetate esters decreased in the order phenyl > o-nitrophenyl > p-nitrophenyl. The simplest explanation of the results is that nitro-substitution causes a decrease in the acylation rate constant ( k 2 ) and this is

v(~IW s- I)

(M)

usually accompanied by a decrease in the dissociation constant of the enzyme-substrate complex QK?). Thus Ks must decrease by a factor of 20 for the sheep enzyme in going from phenyl butyrate to 2,4-dinitrophenyl butyrate. That the trend in k,-,,'s for a series of activated esters is the reverse of that observed for the corresponding alkaline rate constants ( k o ~ , Table I), clearly indicates the participation of factors additional to those which account for the rates of base-catalyzed hydrolysis. In the present work it is not possible to decide whether the size and position of the nitro group account for the observed changes in k,,t and Kn,. However, Malhotra and Philip (21) have shown that substitution of the nitro group by a methyl or a methoxy group does not cause a decrease in k,,t and K,, con~paredwith the corresponding values for phenyl butyrate. Thus for the goat intestinal esterase the steric effect of the nitro group is presumably not large and the reduction in k,,t and K,, must be explained in a different manner. The data of Table 3 show that the enzymes all exhibit some specificity with respect to the chain length of' the acyl group in a homologous series of phenyl esters. Value of k,.,t for the horse, sheep, and pig enzymes increase as the number of carbon atoms in the chain increases from two

-

STOOPS ET

AL. : CARBOXYLESTERASES.


to five. In view of the decrease in alkaline rate constant OH, Table 3) for esters of increasing acyl chain length, the increase in kcat must be attributed to some aspect of specificity which is common to all the carboxylesterases studied. The high kcat's for phenyl formate are not inconsistent with this suggestion, since k,,, (phenyl formate) /' k,,t (phenyl acetate) is 10 for the sheep enzyme, while the ratio of the corresponding alkaline rate constants is about 350. W a l h o t r a and Philip (21) observed a similar trend in kcst's for p-nitrophenyl esters of n-alkyl aliphatic acids from acetate to butyrate, and Ocken and Levy (7) found that kcatfor the pig liver carboxylesterase-catalyzed hydrolysis of the corresponding ethyl esters increased from acetate to valerate. Webb (23) observed a 14-fold increase in the activity of horse liver carboxylesterase towards ethyl esters of the same series from acetate to valerate. Nsn- Miclzuelis-men ten Kiiaetics At least one instance of substrate inhibition has been observed for all of the carboxylesterases studied (Table 5). The sheep enzyme appears to be more susceptible to inhibition by substrate than are the other esterases, although this comparison can be extended only to those enzymes whose kinetic behavior has been examined in the absence of acetonitrile, since high concentrations sf acetonitrile m y inhibit the enzyme, and thus blanket substrate inhibition (compare Tables 1 and 2). Murray (10) observed a substrate inhibition of sheep liver carboxylesterase by ethyl butyrate, and obtained a KI of 9.7 X 10-3 M, in g o d agreement with our estimated value of 7.3 X 1 0 - W (Kt,, Table 5). The rate of sheep liver carboxylesterasecatalyzed hydrolysis of phenyl butyrate at a substrate concentration which results in appreciable substrate inhibition (1.9 mM) is proportional to the enzyme concentration over a 37-fold range. This indicates that inhibition of high concentrations of substrate does not result from a substrate-induced association or dissociation of the enzyme. sDirect comparison of the results with pheilyl formate with the other results is not possible because of the presence of 1.53'1, CHaCN in the former case. However, the effect of 1.535, CCH3CNon the values of kcat is most unlikely to change the argument (cJ values for phenyl butyrate in the absence of CH3CN and the presence of 12.8% CCHaCN (Tables 2, 3)).

IV

57 1

That the same applies to the activation of pig liver carboxylesterase by phenyl butyrate, benzene, and rn-earboxyphenyl n-heptanoate has been conclusively demonstrated (5, 6). Both groups of workers showed that virtually complete dissociation of the enzyme did not result in a decrease in the observed activation by substrate. Further, it was shown that 10mM benzene. which activates pig liver carboxylesterase to the same extent as phenyl butyrate at high concentrations, did not induce a change in the state of aggregation of the enzyme within the time taken to perform kinetic experiments (5). No evidence has been obtained in this laboratory to support the conclusion of Heymann eb ak. (24) that the kinetic behavior of pig liver carboxylesterase is a result of the presence od more than one type of enzyme molecule in the preparation. Stoops et al. (5) proposed the following scheme to describe substrate activation of pig liver carboxylesterase,

in which the modifier- and substrate-activated complexes (MES and SES) decompose with the same rate constant kq, which is larger than kl. As Barker and Jencks (6) have indicated, their results, and those of Ocken and Levy (7) for the pig liver carboxylesterase-catalyzed hydrolysis of ethyl butyrate, are largely consistent with this scheme. It is apparent that Scheme 1, which adequately describes inhibition of the carboxylesterases by substrate, is a formal modification of Scheme 4, achieved by putting k2 = 0. The kinetic behavior of horse liver carboxylesterase at low concentrations of methyl and ethyl butyrates is also consistent with Scheme 4, if it is assumed that K, >> Kc, and the first molecule of substrate may act only as a modifier. Under these conditions, Scheme 4 becomes identical to Scheme 2 (see Results). If, however, [SIowere much greater


572

CAN. J.

BIOCHEM. VBL. 53,

than Kc at the lowest substrate concentration investigated, Michaelis-Menten kinetics would be observed over the experimental substrate concentration range. This possibility was suggested by Barker and Jencks (61, to explain the absence of any observable substrate activation of pig liver carboxylesterase by some substrates. The first molecule of methyl or ethyl butyrate which binds to the horse enzyme acts only as a modifier, and this fact lends further support to their proposal. It may apply equally well to other carboxylesterases which exhibit Michaelis-Menten kinetics. Some comment is necessary on the values of Kc, shown in Table 6. Precise measurensents of initial rates in this low [S] range are difficult. and are very sensitive to the concentration of SES present throughout a kinetic run ([%lo5 Kc). This is probably the major reason for the variation in Kc.which is found for ethyl butyrate. The data for methyl butyrate provide better support for the constancy of Kc., and thus justify the kinetic analysis. The general scheme, therefore, accounts for the apparently varied kinetic results obtained for this group of carboxylesterases. It implies the existence of a modifier site which is presumably close to the active site, and which may bind either a second molecule of substrate or some other small organic molecule. Occupancy of the modifier sits by substrate may produce either an increase in the activity of the enzyme towards that substrate (Scheme 4, path A), or be responsible for the catalytic activity of the enzyme towards a substrate (Scheme 2). Occupancy of this same site by a modifier (other than substrate) may produce an increase in the activity of the enzyme (Scheme 4, path B; Stoops et a%.(5)). However, it is not possible to determine from the present experiments whether the site responsible for the inhibition of these enzymes by substrate (Scheme 1) is the nlodifier site. Nor is it possible to determine from the present data which of the catalytic steps (acylation and/or deacylation) is affected by occupancy of the modifier site, nor whether & is altered under these conditions. As shown in Table 4, the chicken and horse Eiver carboxylesterases exhibit very similar behavior in the catalysis of hydrolysis of phenyl butyrate: kcat shows a clean dependence on a pKR' of 5.8 for the chicken enzyme and on a

1975

pKaf of 4.75 for the horse enzyme. Further, both enzymes exhibit an increase-in K,,, for phenyl butyrate in the pH range from 3 to 6. Similarly, Stoops et ~61.(5) obtained pKaHs of 4.$ and 5.1 for the hydrollysis of phenyl and p-nitrophenyl butyrates, respectively, in the presence of 12.8YG acetonitrile (pig liver enzyme), and Adler and Kistiakowsky (4) observed a pKItl of 4.7 for the hydrolysis of methyl beatyrate (horse liver enzyme). In all of the above examples, MichaelisMenten kinetics were observed. In systems where substrate activation is observed, the pH dependence of appears to be more complex. Studies by Bcken and Levy (7) indicate, in terms of scheme 4. that kl and k2 exhibit different pH dependencies (pKZr's of 5.8 and 3.8, respectively) for the hydrolysis of ethyl butyrate catalyzed by the pig liver enzyme. Similar results were obtained by Adler and Kistiakowsky (4)Since kc,t-pH profiles obtained in the present work correspond exactly to theoretical titration curves, it is likely that they represent the ionization of a single catalytic group in each enzyme. However, a definite assignment of the reported pKaf's to particular residues is not possible at this stage. Work in this laboratory on the inhibition of the earboxylesterases by bromoacetsphenone (25) and by the hydroraylamine - cupric ion oxygen system (Runnegar et a?. (26); Keto, A. I., and Zerner, B. : unpublished results) strongly implicates the participation of histidine in the mechanism of action of these enzymes. Further studies will be directed t ~ ~ a a~positive d s identification of' the catalytically important groups at the active sites of the carboxylesterases, and towards an understanding of the manner in which particular modifiers affect the active site. We wish to thank Professor Myron Bender for permission to use tinpublished data from the Ph.B. thesis of J . K . S. 1 . Augusteyn, R. C . , de Jersey, J., Webb, E. C. & Zerner, B. (1969) Bicrchim. Biopltj~s.Acfa 171,128-139 2. Scott. M. & Zerner, B. (1975) C u i ~ J. . Biocl~era?.93, 561-564 3. Mrisch, K. (197 1) Eitzyrates 5 , 43-69 4 . Adier, A. J. $r Kistiakswsky, G . B. (1962) J. Am. Chem. Soc. 84, 695-703 5. Stoops, J . K . , Hsrgan, D. J., Warnnegar, M. T. C., de Jersey, J., Webb, E. C. & Zera~er.B. (1959) Biochemistry 8, 2026-2033 6. Barker, D. L. Bs Jencks, W. P. ( 1969) Biochernistrg, 8 , 390-3897

STOOPS ET AL. : CAWBOXYLESTERASES. I V


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Carboxylesterases from pig and ox liver.

Carboxylesterases (EC 3.1.1). The source of variations in substrate specificity and properties of pig liver carboxylesterase.

Heterogeneity of carboxylesterases in rat liver cells.

Comparison of substrate specificity among human arylacetamide deacetylase and carboxylesterases.

Hydrolysis of pyrrolizidine alkaloids by guinea pig hepatic carboxylesterases.

Carboxylesterases: General detoxifying enzymes.

Synthetic cannabimimetic agents metabolized by carboxylesterases.

Regulations of Xenobiotics and Endobiotics on Carboxylesterases: A Comprehensive Review.

A new glycopeptide in pig, ox and sheep pituitary.

The catalytic mechanism of carboxylesterases: a computational study.

Topogenesis of carboxylesterases: a rat liver isoenzyme ending in -HTEHT-COOH is a secreted protein.

Purification and characterization of carboxylesterases from rat lung.

Characterization of molecular species of liver microsomal carboxylesterases of several animal species and humans.

Carboxylesterases: Dual roles in lipid and pesticide metabolism.

Kinetic properties of aldehyde dehydrogenase from sheep liver mitochondria.

Crystal structures of two Bacillus carboxylesterases with different enantioselectivities.

Selective stimulation of carboxylesterases metabolizing charged steroid esters by hydrocortisone.

Is histidine involved in the catalytic mechanism of unspecific carboxylesterases?

Screening of specific inhibitors for human carboxylesterases or arylacetamide deacetylase.

Some properties of aldehyde dehydrogenase from sheep liver mitochondria.

Some properties of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver.

Organophosphorus flame retardants inhibit specific liver carboxylesterases and cause serum hypertriglyceridemia.

Carboxylesterases in guinea pig. A comparison of the different isoenzymes with regard to inhibition by organophosphorus compounds in vivo and in vitro.

Carboxylesterases with defferent substrate specificity in human brain extracts.