acylenzyme mechanism and solvent isotope effects for cholesterol esterase-catalyzed hydrolysis of...

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6 BBA 52027 Acylenzyme mechanism aud solvent isotope effects for cholesterol e&erase-catalyzed hydrolysis of p-nitrophenyl butyrate Jay S. Stout, Larry D. Sutton and Daniel M. Quinn * (Received March 12th. 1985) (Revised manuscript received July 16th. 19X5) Key words: Cholesterol &erase; Acylenzyme mechanism; Solvent isotope effect: Proton transfer; Transition state The m~hanism of cholesterol &erase- (carboxylic ester hydrolase, EC 8.1.1.1) catalyzed hydrolysis of the water-soluble ester p-nitrophenyl butyrate has been characterized for commercially available preparations from bovine and porcine pancreas and for a purified preparation from porcine pancreas. Kinetic evidence for an acylenzyme mechanism is provided by experiments wherein the butyryl enzyme is trapped by MeOH, EtOH or n-BuOH. For the last alcohol the transacylation product n-butyl n-butyrate was characterized by W-mass spectrometry. Solvent isotope effects have been measured for I&,,/ K,, which is the rate constant for acylation, and for Vm,,, which monitors rate-dete~ining deacylation. Isotope effects of 1.5-3 on these rate constants indicate that both steps of the acylenzyme m~hanism for cholesterol esterase catalysis involve transition states that are stabilized by general acid-base proton bridges. Pancreatic cholesterol esterase is released into the duodenum in response to an alimentary fat load, where it catalyzes the hydrolysis of acylglycerols, cholesteryl esters and phospholipids [l--3]. The presence of cholesterol esterase activity in the intestinal tract is necessary for the full absorption of dietary cholesterol into the bloodstream [4,5]. When the activity of the enzyme is blocked, adsorption of cholesterol drops by 80% 151. Because of the correlation between serum cholesterol levels and atherosclerosis [6], the devel- opment of methods for inhibiting cholesterol esterase activity is a desirable goal. The design of mechanism-based inhibitors should be greatly * To whom correspondence should be addressed. Abbreviations: MeOH. methanol: EtOH. ethanol; n-BuOH. n-butanol; PNPB. p-nitrophenyt butyrate; LBTI-agarose. lima bean trypsin inhibitor-agarose; GC. gas chromatography. facilitated when the mechanism of cholesterol esterase catalysis has been defined. Lombard0 and Guy [7] have characterized the effects of the nucleophiles methanol and butanol on human pancreatic cholesterol esterase turnover of p-nitrophenyl acetate and n-propylthiol acetate. They interpret their results in terms of partitionin of an acetylenzyme intermediate between alcohol- ysis and hydrolysis. In addition. chemical modifi- cation experiments suggest that serine and histi- dine are components of the cholesterol esterase active site [8] and therefore that cholesterol esterase catalysis follows a serine hydrolase mechanism. In this paper we extend Lombard0 and Guy’s mecha- nism to bovine and porcine pancreatic cholesterol esterase-catalyzed hydrolysis of p-nitrophenyl butyrate (PNPB) by using MeOH, EtOH and tt- BuOH as nucleophiles. We have also measured solvent deuterium kinetic isotope effects for the acylation and deacylation stages of cholesterol esterase-catalyzed hydrolysis of PNPB. Our results BUS-276~/~5/$03.30 ‘1:. 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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BBA 52027

Acylenzyme mechanism aud solvent isotope effects for cholesterol

e&erase-catalyzed hydrolysis of p-nitrophenyl butyrate

Jay S. Stout, Larry D. Sutton and Daniel M. Quinn *

(Received March 12th. 1985)

(Revised manuscript received July 16th. 19X5)

Key words: Cholesterol &erase; Acylenzyme mechanism; Solvent isotope effect: Proton transfer; Transition state

The m~hanism of cholesterol &erase- (carboxylic ester hydrolase, EC 8.1.1.1) catalyzed hydrolysis of the water-soluble ester p-nitrophenyl butyrate has been characterized for commercially available preparations from bovine and porcine pancreas and for a purified preparation from porcine pancreas. Kinetic evidence for an acylenzyme mechanism is provided by experiments wherein the butyryl enzyme is trapped by MeOH, EtOH or n-BuOH. For the last alcohol the transacylation product n-butyl n-butyrate was characterized by W-mass spectrometry. Solvent isotope effects have been measured for I&,,/ K,, which is the rate constant for acylation, and for Vm,,, which monitors rate-dete~ining deacylation. Isotope effects of 1.5-3 on these rate constants indicate that both steps of the acylenzyme m~hanism for cholesterol esterase catalysis involve transition states that are stabilized by general acid-base proton bridges.

Pancreatic cholesterol esterase is released into the duodenum in response to an alimentary fat

load, where it catalyzes the hydrolysis of acylglycerols, cholesteryl esters and phospholipids [l--3]. The presence of cholesterol esterase activity in the intestinal tract is necessary for the full

absorption of dietary cholesterol into the

bloodstream [4,5]. When the activity of the enzyme is blocked, adsorption of cholesterol drops by 80%

151. Because of the correlation between serum cholesterol levels and atherosclerosis [6], the devel- opment of methods for inhibiting cholesterol esterase activity is a desirable goal. The design of mechanism-based inhibitors should be greatly

* To whom correspondence should be addressed.

Abbreviations: MeOH. methanol: EtOH. ethanol; n-BuOH.

n-butanol; PNPB. p-nitrophenyt butyrate; LBTI-agarose. lima bean trypsin inhibitor-agarose; GC. gas chromatography.

facilitated when the mechanism of cholesterol esterase catalysis has been defined.

Lombard0 and Guy [7] have characterized the

effects of the nucleophiles methanol and butanol on human pancreatic cholesterol esterase turnover

of p-nitrophenyl acetate and n-propylthiol acetate. They interpret their results in terms of partitionin

of an acetylenzyme intermediate between alcohol- ysis and hydrolysis. In addition. chemical modifi- cation experiments suggest that serine and histi-

dine are components of the cholesterol esterase

active site [8] and therefore that cholesterol esterase catalysis follows a serine hydrolase mechanism. In

this paper we extend Lombard0 and Guy’s mecha- nism to bovine and porcine pancreatic cholesterol esterase-catalyzed hydrolysis of p-nitrophenyl butyrate (PNPB) by using MeOH, EtOH and tt- BuOH as nucleophiles. We have also measured solvent deuterium kinetic isotope effects for the acylation and deacylation stages of cholesterol esterase-catalyzed hydrolysis of PNPB. Our results

BUS-276~/~5/$03.30 ‘1:. 1985 Elsevier Science Publishers B.V. (Biomedical Division)

are interpreted in terms of a cholesterol esterase mechanism that resembles the mechanism of serine proteinase catalysis [9].

Materials and Methods

Materials

Bovine and porcine pancreatic cholesterol esterase and PNPB were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). For some experiments, potential trypsin impurities were removed from the commercial bovine pancreatic cholesterol esterase preparation by affinity absorp- tion on LBTl-agarose [lo]. A purified preparation of porcine pancreatic cholesterol esterase was sup- plied gratis by Dr. Howard Brockman of Hormel Institute, Austin, MN. Deuterium oxide (99.8 atom% ‘H) was purchased from Aldrich Chemical Co. (U.S.A.) and was used as received. MeOH (Mallinckrodt, U.S.A.) and 95% EtOH (Matheson, Coleman and Bell Co., U.S.A.) were used without further purification; n-BuOH (Fisher Scientific Co., U.S.A.) was fractionally distilled before use. All other materials were commercially-available reagent grade chemicals.

Enzyme kinetics

Cholesterol esterase-catalyzed hydrolysis of PNPB was monitored by following production of p-nitrophenoxide at 400 or 450 nm on a Beckman DU7 UV-visible spectrophotometer that was inter- faced to an IBM Personal Computer. Reactions were performed in buffer (pH values and buffer compositions are given in figure legends) at 25.0 + O.l”C. V,,, was sometimes determined by the method of initial rates at [PNPB], > K,. Initial rates were calculated by linear least-squares analy- sis of less than 5% of time courses for total sub- strate turnover. For reactions in which [PNPB], -=+K K,, first order rate constants equal k’&,,,/K, and are calculated by nonlinear least-squares fit to the three parameter exponential function:

A = (A,, - A,) ,-~~m.J~r”)~ + A, (1)

In Eqn. 1, A, A, and A, are absorbances at times t, 0 and co, respectively.

For reactions in which [PNPB], = l-5 K,, time courses for total substrate turnover are described

by the integrated form of the Michaelis-Menten equation:

Eqns. 3 can be used to substitute for substrate concentrations in terms of absorbances.

(3)

If one makes this substitution and divides both sides of the equation by A - A,, one gets Eqn. 4:

In k--A, I K =m 4-A I I

A- 41 Lx A-A, +f/max~+\ (4)

Eqn. 4 can be converted into a form that is similar to the Lineweaver-Burk equation by writing an Eqn. 4 at t = i and at t = i + 10 and by subtract- ing the former from the latter:

In A, -4

I ,+10-f, K, A,-A,+,o 1 1

A -4 Vma, A,+to-A, +c~p-fs (5) ,+1*

The absorptivity constant at 400 and 450 nm of the reactant PNPB, E,, is 0. That of the product, p-nitrophenol plus p-nitrophenoxide, is calculated at the experimental pH by using the absorptivity constant of p-nitrophenoxide of 21388 at 400 nm or 4135 at 450 nm and the pK, = 6.98 of p- nitrophenol. The term on the left-hand side of Eqn. 5 is a reciprocal velocity (i.e., dt/d A) in t. A-’ units, while the absorbance function on the right-hand side is directly proportional to [S]- i. In a typical experiment 300 { t, A} pairs are acquired by the IBM Personal Computer from the spectro- photometer at l-s intervals. The respective terms of Eqn. 5 are calculated by using absorbance readings at t = 0 and t = 10 s, at t = 1 and t = 11 s, etc., until 97% of the reaction is reached. The calculated double-reciprocal array is then fit by weighted linear least-squares to Eqn. 5, using weighting factors that are proportional to (d A/dt)4 [ll]. The linear least-squares analysis allows calculation of V,,, and K, in units A 1 s- ’ and A, respectively. The corresponding parame-

8

ters in M. s-l and M units are obtained by divid- ing by Ed.

The pH values of buffer solutions used for the determination of the enzyme kinetics were mea-

sured with a Corning Model 125 pH meter

equipped with a glass combination electrode. For

deuterium oxide buffers, 0.4 was added to the pH

meter reading [12].

TABLE I

SOLVENT ISOTOPE EFFECTS FOR CHOLESTEROL

ESTERASE-CATALYZED HYDROLYSIS OF PNPB

Runs were done at 25.0~0.1’C in 1.04 ml of buffer. “‘t’=

I’,“,$‘/ <,ii;O and +?P/K) = (V,,;,,/K,,)t'2"/(CI",~\,/ K,,, jH~". Error limits of isotope effects were calculated accord- ingto: 3(~Hli)=1Hk[(~kH10,kHi”)’ +(~~‘H’O,k’HIO)?]I 2.

Enzyme source -H(P/K) -ii p

Nucleophilic activation and product analysis

Addition of alcohols (MeOH, EtOH or n-

BuOH) to an assay for cholesterol esterase-cata-

lyzed hydrolysis of PNPB increases both V,,, and K,, which is consistent with nucleophilic trapping

of a butyryl cholesterol esterase intermediate (see

Results for analysis of activation data). Hence,

chemical characterization of the predicted trans-

acylation product was undertaken when n-BuOH was the nucleophile. To 100 ml of 0.05 M trizma buffer (pH 7.82) that contained 0.1 M NaCl, 30 pg

cholesterol esterase from bovine pancreas and 2% n-BuOH (v/v) was added, with stirring, 1.0 ml of

0.2 M PNPB in CH,CN. After sufficient time for

total substrate turnover had elapsed, successive

additions of PNPB in CH,CN were made for a total of four additions. While the reaction pro-

ceeded, a sweet-smelling liquid separated and

floated on the aqueous buffer. The sweet-smelling

liquid was extracted into 20 ml of diethyl ether

and the diethyl ether layer was washed with 2% NaHCO, and with distilled water. After drying

with CaSO, the diethyl ether layer was analyzed on a Hewlitt-Packard 5985B GC-mass spectrome- ter that was operated in the electron ionization mode at 70 eV. The identity of the major GC peak

was verified by computerized matching with mass

spectra that are contained in the NIH/EPA/ MSDC mass spectral data base. Similar product

analyses were undertaken when transacylation was catalyzed by purified porcine pancreatic cholesterol esterase.

Deuterium isotope effects

Solvent deuterium kinetic isotope effects were determined by measuring rate constants in buffered H,O and in equivalent buffers in ’ H,O. Equiv- alent H,O and 2H,0 buffers are those that con- tain the same concentrations of acid and base components of the buffer pair [13,14]. The particu-

Bovine pancreas (Sigma) 1.49 + 0.06 .’ 1.91 + 0.06 ’

Bovine pancreas (Sigma) ’ 1.8 +O.l 2.51 kO.08

Porcine pancreas (Sigma) ’ 1.8 kO.2 2.4 kO.2

Porcine pancreas (purified) ’ 1.5 Ifro.3 2.9 +0.4

” Reactions contained 58 pg/ml protein and [PNPB],, = 3.8

FM ( = K,,/33) in 0.01 M Tris buffer (pH 8.09 or p’H 8.70)

that contained 0.1 M NaCI. Rate constants were calculated

by using Eqn. 1.

h Reactions contained 7.2 yg/ml protein and [PNPB],, = 0.58

mM ( = 4.5 K,,) in the buffers of footnote a. Rate constants

were determined by the method of initial rates.

’ Cholesterol esterase preparation was treated with LBTI-

agarose to remove trypsin. Reactions contained 5.8 pg/ml

protein and [PNPB], = 0.19 mM ( = 0.9 K,,) in 0.1 M sodium

phosphate buffer (pH 7.39 or p’H 7.96) that contained 0.1 M

NaCl. Rate constants were calculated by using Eqn. 5.

’ Reactions contained 65 pg/ml protein and [PNPB],, = 0.20

mM ( = 2.1 K,) in 0.1 M sodium phosphate buffer (pH 7.32

or p’H 7.82) that contained 0.1 M NaCI. Rate constants were

calculated by using Eqn. 5.

’ Reactions contained 3.8 pg/ml cholesterol esterase and

[PNPB], = 0.37 mM ( = 0.94 K,) in 0.1 M sodium phos-

phate buffer (pH 7.41 or p’H 7.97) that contained 0.1 M

NaCI. Rate constants were calculated by using Eqn. 5.

lar buffers used and their pH or pZH are described in Table I.

Results

Activation by alcohols Except at the highest alcohol concentrations,

addition of alcohols to cholesterol esterase-cata- lyzed hydrolysis of PNPB was accompanied by percent increases in V,,, and K, that are similar in magnitude. At low concentrations of EtOH and n-BuOH the intercepts of Lineweaver-Burk plots decrease but the slopes are unchanged, as Fig. 1 shows for the addition of n-BuOH. This kinetic pattern can be rationalized in terms of the acyl-

Michaelis-Menten parameters:

I OO

t I t t I I

4 8 12

fn(Absorbance), Abs-’

Fig. 1. Activation of cholesterol esterase-catalyzed hydrolysis of PNPB by n-butanol. Plots are weighted fits of time course data to the linear transform of the integrated Michaelis-Menten equation (Eqn. 5). The y-coordinate of the plot corresponds to the left-hand side and the x-coordinate (function of ab- sorbance) of the plot to the composite absorbance term on the right-hand side of Eqn. 5, respectively. Each run was done at 2S.0+0.1°C in 1.04 ml of 0.05 M t&ma buffer (pH 7.74) that contained 0.1 M NaCI, 1.9% MeCN (v/v). 30 pg bovine pancreatic cholesterol esterase, [PNPB], = 0.2 mM, and no n-BuOH (A), 0.04 molal n-BuOH (0) or 0.33 molal n-BuOH (e). The kinetic parameters in the absence of n-BuOH are K,=52+2 FM and 0,,,=4.2+0.3~M.s-‘.

enzyme mechanism of Scheme I:

*1 z *-1

hcmnzym

Scheme I. Mechanism for cholesterol esterase-catalyzed hydrol- ysis and transacylation of PNPB.

In Scheme I Ser and Im represent the active site serine and imidazole (of histidine) side-chains that Lombard0 [S] proposes for human pancreatic cholesterol esterase. C3H,C0,pNP is PNPB and “OpNP is the p-nitrophenoxide product. Proton transfer, which is a proposed molecular dynamic element of choiesterol esterase catalysis in Scheme I, will be discussed later.

For the mechanism of Scheme I a steady-state derivation gives the following predictions for the

In these equations KS = (k_, + k,)/k,, [El, is the cholesterol esterase concentration and [Nu] is the concentration of the added nucleophilic alcohol. Eqns. 5 predict equal changes in V,,,, and K, when alcohols nucleophilically trap butyryl- cholesterol esterase. V,,,/K, does not change, however, and hence the slope of the Lineweaver- Burk plots does not change. This is just the kinetic pattern observed at low concentrations of EtOH and n-&OH.

Fig. 2 shows plots of the percent increases of the Michaelis-Menten parameters effected by ad- dition of MeOH, EtOH and n-BuOH to pan- creatic cholesterol esterase-catalyzed hydrolyses of PNPB. The percent increases for K, and I&,, are similar in magnitude except at the two highest concentrations of EtOH and n-BuOH, where K, increases more rapidly with increasing nucleophile concentration than does V,,,. This is likely due to competitive inhibition of the reaction at high al- cohol concentrations. The Lineweaver-Burk plots of Fig. 1 for the two higher concentrations of n-BuOH show a pronounced slope effect, which is consistent with the onset of competitive inhibition. Nonetheless, at the lower alcohol concentrations the similar increases in K, and V,,,, signal nucleophilic trapping of a butyryl cholesterol esterase intermediate, as the parallel Lineweaver- Burk plots of Fig. 1 also indicate. Fig. 2B contains data for the effect of n-BuOH on bovine and porcine cholesterol esterase-catalyzed hydrolyses of PNPB. Two sources of porcine cholesterol esterase were used, enzyme from Sigma Chemical Co. and purified enzyme provided by Dr. Howard Brockman. These two cholesterol esterase prepara- tions showed percent increases in K, and V,,, that were equal within experimental error, and therefore the mean percent changes are plotted in Fig. 2B. For the nucleophiles tested the order of efficacy of activation is n-BuOH = EtOH > MeOH.

In order to verify that activation of cholesterol

10

0- 0 1.0 2.0 3.0 4.0

1600

[ROH], molal

B

0” I I I

0 0.1 0.2 0.3

[n-BuOH], molal

Fig. 2. Effect of alcohols on the Michaelis-Menten parameters

for cholesterol esterase catalyzed hydrolysis of PNPB. (A)

Activation by MeOH and EtOH. Reactions were done under

the conditions listed in the Fig. 1 legend, save that they

contained the indicated concentrations of MeOH or EtOH and

no n-BuOH. Closed symbols, K, activation; open symbols.

V,,,, activation; squares. EtOH activation data; circles. MeOH

activation data. (B) Activation by n-BuOH. Closed symbols,

K, activation; open symbols. V,,,x activation; circles, bovine

cholesterol esterase activation; squares, porcine cholesterol

esterase activation. Reactions were done under the conditions

listed in the Fig. 1 legend. save that [n-BuOH] was varied as

indicated and the following concentrations were used: bovine

cholesterol esterase reactions contained [PNPB],, = 0.4 mM at

the three lower [n-BuOH] levels and [PNPB], = 0.8 mM at the

highest [ n-BuOH]. Porcine cholesterol esterase reactions con-

tained 2 pg protein and [PNPB], = 0.6 mM (purified enzyme)

or 0.27 mg protein and [PNPB],, = 0.4 mM (Sigma enzyme).

The kinetic parameters in the absence of n-BuOH were K, =

73+3 PM, V,,,,x = 9.OkO.9 PM’S ’ (purified enzyme) and

K,= 0.12kO.02 mM. I$,,, = 5.1750.35 pM.s~ ’ (Sigma en-

zyme).

esterase by alcohols occurs via a nucleophilic

mechanism, n-butyl n-butyrate that is formed dur-

ing bovine pancreatic cholesterol esterase turnover

of PNPB in the presence of n-butanol was isolated

and characterized by GC-mass spectrometry. The

major peaks of the mass spectrum and corre-

sponding fragment assignments are given in Scheme II:

m/e- 89 m/e = 101 m/e- 116

Scheme II. Mass spectral peaks for product of cholesterol

esterase-catalyzed transacylation.

The fragment assignments are consistent with n-

butyl n-butyrate as the transacylated product. which is supported by the computer matching of our mass spectrum with that of n-butyl n-butyrate that is contained in a mass spectral data base of

about 26000 compounds (cf., Material and Meth- ods). Similar results were obtained when purified

porcine pancreatic cholesterol esterase was the transacylation catalyst.

Sohent isotope effects

The measurement of solvent deuterium kinetic

isotope effects is an often used technique for dem-

onstrating the existence in enzyme reactions of

general acid-base transition state proton bridges [13.14]. Table I contains solvent isotope effects for

Yl,,, (rate-determining deacylation) and for

V,,,/K, (rate-determining acylation) of various cholesterol esterase-catalyzed hydrolyses of PNPB. The isotope effects for both steps are sizeable, which suggests that proton transfers are dynamic elements of cholesterol esterase mechanism and transition state structure. Treatment of bovine pancreatic cholesterol esterase with LBTI-agarose to remove trypsin resulted in a preparation that gave somewhat higher -isotope effects than the untreated preparation. However, since the solvent isotope effects of Table I are sizeable throughout, these differences do not compromise the conclu- sions concerning the mechanism of cholesterol

11

esterase catalysis presented in the following Dis-

cussion.

Discussion

In this paper we present kinetic evidence for an acylenzyme mechanism for bovine and porcine pancreatic cholesterol esterase catalyses. Addition

of alcohols activates cholesterol esterase-catalyzed

turnover of PNPB, which is due to nucleophilic

trapping of a butyryl cholesterol esterase inter- mediate (cf., Scheme I). Furthermore, nucleophilic

activation by alcohols necessitates that, in the ab-

sence of nucleophiles other than water, deacylation

is the rate-determining step that I$,,, monitors.

This can be demonstrated by considering the form

of Eqns. 6 when [Nu] = 0:

If deacylation is rate-determining (i.e., k, -=z k,),

V,,, = k,[E], and addition of nucleophiles will increase V,,, by providing a parallel pathway

k,[Ntij- for deacylation. If acylation is rate-de-

termining (i.e., k, B- k,), V,,, = k,[E], and nucleophiles cannot affect the reaction rate. Hence, V,,, always monitors deacylation. Similar conclu-

sions were reached by Lombard0 and Guy [7] for

nucleophilic activation of human pancreatic

cholesterol esterase-catalyzed turnover of acetate

esters. On the other hand, V&,/K,,, = k,[E],/K,

and therefore always monitors rate-determining

acylation. This result is consistent with the general

assertion that V_,&K, always monitors the rate-

determining step through the first irreversible event

of the catalytic mechanism [15]. For cholesterol esterase turnover of PNPB, the first irreversible event is the release of p-nitrophenoxide which

completes the acylation stage of the reaction. The acylation and deacylation stages of cholesterol esterase-catalyzed hydrolysis of PNPB can thus be separated kinetically.

Lombard0 showed by chemical modifications that serine and histidine are likely constituents of the active site of human pancreatic cholesterol esterase [8]. Those results, along with the evidence for acylenzyme intermediates presented herein and by Lombard0 and Guy [7], leads us to predict that

cholesterol esterase catalysis occurs via a mecha-

nism like that of the serine proteinases [9,15]. As Scheme I outlines, this mechanism involves proton transfers in both the acylation and deacylation

stages of catalysis. That proton transfers are

structural features of transition states of cholesterol esterase catalysis is supported by the data of Table

I. Solvent isotope effects of 1.5-1.8 for V,,,/K,

(acylation) and of 1.9-2.9 for V,,, (deacylation) are similar in magnitude to those frequently ob-

served for serine protease catalysis [13-151. The cholesterol esterase reactions for which

kinetic investigations indicate an acylenzyme

mechanism involve the use of water-soluble sub-

strates. The same holds true for the solvent isotope

effects discussed herein. However, physiological

cholesterol esterase catalysis is heterogeneous bio-

catalysis, wherein lipid substrate turnover occurs

at lipid-water interfaces. It remains to be seen

whether interfacial cholesterol esterase catalysis is

characterized by acylenzyme kinetics and sizeable

solvent isotope effects. Substrate systems to probe these aspects of interfacial cholesterol esterase

catalysis are being developed in our laboratory.

Acknowledgements

The authors thank Dr. Howard Brockman of

Hormel Institute for providing a purified prepara-

tion of porcine pancreatic cholesterol esterase, Dr. David Wiemer for helpful discussion and assis-

tance with mass spectrometry, and Ms. Pat Spencer for technical assistance. This research was sup-

ported by American Heart Association Grant-In- Aid 84 1003.

References

1 Brockerhoff, H. and Jensen, R.G. (1974) Lipolytic En-

zymes, pp. 176-193, Academic Press, New York

2 Kritchevsky, D. and Kothari, H.V. (1978) Adv. Lipid Res.

16, 221-266

3 Rudd. E.A. and Brockman, H.L. (1984) in Lipases

(Burgstrom, B. and Brockman, H.L., eds.). pp. 185-204,

Elsevier, Amsterdam 4 Bhat, S.G. and Brockman, H.L. (1982) Biochem. Biophys.

Res. Commun. 109, 486-492

5 Gallo, L.L., Clark. S.B., Myers. S. and Vahouny, G.V.

(1984) J. Lipid Res. 25, 604-612

6 Kannel, W.B., Castelli, W.P., Gordon, T. and McNamara,

P.M. (1971) Ann. Int. Med. 74, l-12

12

7 Lombardo, D. and Guy, 0. (1981) B&him. Biophys. Acta

657. 425-437

8 Lombardo, D. (1982) Biochim. Biophys. Acta 700, 67-74

9 Stroud, R.M. (1974) Sci. Am. 231. 74-88

10 Lynn, K.R.. Chuaqui. C.A. and Clevette-Radford. N.A.

(1982) Bioorganic Chemistry 11, 19-23

11 Cleland, W.W. (1967) Adv. Enzymol. Relat. Areas Mol.

Biol. 29, l-32

12 Salomaa. P.. Schaleger, L.L. and Long, F.A. (1964) J. Am.

Chem. Sot. 86. l-7

13 Schowen, K.B.J. (1978) in Transition States of Biochemical

Processes (Gandour. R.D. and Schowen, R.L.. eds.), pp. 2255283. Plenum Press, New York

14 Schowen. K.B. and Schowen, R.L. (1982) Methods En-

zymol. X7, 551-606

15 Hegazi, M.F.. Quinn. D.M. and Schowen. R.L. (1978) tn

Transition States of Biochemical Processes (Gandour. R.D.

and Schowen. R.L.. eds.), pp. 355-428. Plenum Press, New

York