Eur. J. Biochem. 817.439-451 (1978)

Protease I1 from Escherichia coli Substrate Specificity and Kinetic Properties Michele PACAUD Laboratoire de Chimie des Protkines, Institut de Kecherches Scientifiques sur le Cancer, Villejuif (Received July 20. 1977)

Protease 11, a cytoplasmic endopeptidase from Escherichia coli, has been further characterized. In agreement with previous evidence of a trypsin-like specificity, this enzyme cleaves essentially the carboxymethylated B chain of insulin at the Arg2-Gly23bond, but after prolonged periods of incubation it is also able to cleave the T~r’~-Leu’’ bond. Protease I1 and pancreatic trypsin are inhibited in a similar fashion by diisopropylphosphofluoridate and tosyl lysine chloromethyl ketone suggesting that seryl and histidyl groups are commonly involved in the active site of the two enzymes. The analysis of the pH dependence of the steady-state kinetic parameters of two related substrates, N-benzoyl-L-arginine ethyl ester and N-benzoyl-L-arginine p-nitroanilide, indicates that two ionizing groups are present in the enzyme’s active site, one has a pK(app) of 6.7-7.5, the other has a pK(app) of 8.7-9.6. These two enzyme groups control the binding of substrates, but the group with a pK around 9 does not appear to be essential for the catalysis. The assignment of these pK values is discussed in connection with the known features of the reaction mechanism of serine proteases. Attempts to obtain an insight into the catalytic mechanism from the kinetic analysis of the effects of added nucleophiles were unsuccessful. No ‘burst’ release ofp-nitrophenol is observed from the hydrolysis ofp-nitrophenyl-p’-guanidobenzoate. Deuterium isotope effects for k J K , range from 1 for N-benzoyl-L-arginine ethyl ester to 1.5 for N-benzoyl-L-arginine amide. Furthermore, the kinetics of the protease-11-catalyzed hydrolysis of various synthetic substrates are characterized by relatively weak variations in the values of K,(app), but significant differences in the values of k,,, are observed with closely related amide and ester substrates. This implies that K , may be related to K , , and that the step immediately following formation of the Michaelis enzyme-substrate complex in the catalytic process, is rate-determining. These findings do not agree with the acyl-enzyme mechanism established for pancreatic serine hydrolases and cr-lytic protease.

Escherichia coli contains several neutral proteinases [l - 31 some of which appear to be serine proteinases on the basis on their irreversible inhibition by diisopropylphosphofluoridate [2,4]. One of them, a cytoplasmic enzyme named protease 11, has been recently purified and some of its physicochemical properties Abbreaiotions. Dip-F, diisopropylphosphofluoridate;Tos-LysCH,CI. N-tosyl-r-lysine chloromethyl ketone; Rz-Arg-OEt, N benzoyl-~-arginineethyl ester; Bz-Arg-NAn, N-benzoyl-L-argininep-nitroanilide ; Bz-Arg-NH, , N-benzoyl-t-arginine amide ;Tos-ArgOMe. N-tosyl-L-arginine methyl ester; Tos-Lys-OMe. N-tosyl-Llysine methyl ester; Cbz-Lys-ONp, N-benzyloxycarbonyl-L-lysinep-nitrophenyl ester; Cbz-Lys-OBzl, N-benzyloxycarbonyl-L-lysine benzyl ester; Ac-Tyr-OEt, N-acetyl-L-tyrosine ethyl ester; Ac-TyrNAn, N-acetyl-L-tyrosine-p-nitroanilide. Enzymes. Bovine trypsin (EC 3.4.21.4); protease I1 (EC 3.4.21 .-).

have been described [4,5].Like pancreatic trypsin, protease I1 is specific for arginine and lysine derivatives and is inhibited by tosyl lysine chloromethyl ketone, suggesting that the two enzymes may have some analogous kinetic behavior. Nevertheless they differ widely in molecular weight, structural stability, and sensitivity to naturally occurring trypsin inhibitors [4]. Because of the very different phylogenetic sources and physiological functions of protease I1 and pancreatic trypsin, it has appeared of interest to characterize further the Escherichia coli enzyme with respect to its catalytic properties. Inferences about its mechanism have been based both on studies of its substrate catalysis and on analogies drawn from trypsin and chymotrypsin.

Protease 11 from Escherichici c d i

440

The hydrolytic reactions catalyzed by pancreatic serine proteases usually are described by Eqn (1) [6,7]: ECHZOH

+ RCOX

ECH20H :RCOX A HX (Michaelis complex)

+ ECH,OCOR

5RCOH + ECH,OH

(PI) (3)

P Z )

in which CH,OH represents the active serine of the enzyme, and RCOX is an amide or ester substrate. Here k , / k - , gives the binding constant K , for formation of the Michaelis complex; kz and k , are the acylation and deacylation rate constants. The aboveequation represents a minimum sequence of steps in the overall catalytic process, since it has been recently reported that an unstable tetrahedral intermediate is involved in the conversion of the initial Michaelis complex to the species which loses the first product [8, 91. To ascertain whether the kinetics of hydrolysis of small synthetic substrates by protease I1 are consistent with Eqn (l), some properties of the steady-state parameters derived from this equation were examined. The experimental results are reported here along with some studies on the chemical inactivation of the enzyme, and on substrate specificity towards polypeptides of known sequence.

dioxane were high purity products (spectral grade) from Carlo Erba (Milano, Italy); they were stored in black bottles tightly stoppered and used directly. All other chemicals were of the highest purity available. Bufers und Solcunt/Eli.ctrolvte Mixtures The dependence of enzyme activity on pH was measured in the following buffers: 0.1 M sodium phosphate from pH 6 to 7.6, 0.1 M TrisiHCl from pH 7.75 to 8.5 (final ionic strength=O.l M), and 0.1 M ammonium bicarbonate/ammonia from pH 8.6 to 10 (final ionic strength=0.1 M). pH was determined at room temperature (20-25 'C) with a radiometer pH meter model 26, standardized in water pH-6.5 or pH-9 standard buffers. For solutions in 'H,O, the p2H of the solution was calculated from the measured pH by adding 0.40 pH unit [lo]. The solvent/electrolyte mixtures were prepared by weight. The weight of water was calculated from the exact volume and density at the temperature of preparation. The dielectric constants ( D )of the solutions at 25 "C were obtained from plots of log D versus percentage weight of organic compound, as calculated from data reported by Akerlof [ I l l for methanol and isopropanol and by Akerlof and Short [I 21 for dioxane. Isolation of' Protease II

EXPERIMENTAL PROCEDURE Muter irr Is Bovine trypsin was a twice-crystallized preparation from Worthington Biochemical Co. (U.S.A,)Carboxymethylated B chain of porcine insulin, dansyl amino acids, and Tris (ultra pure) were purchased from SchwartziMann (U.S.A.) Luteinizing hormone releasing factor and N-benzoyl-L-arginine-p-nitroanilide were obtained from Bachem. Inc. (Marina Del Rey, Cal.. U. S.A. ). p-Nitrophenyl-p'-guanidobenzoate .HCI and N-benzyloxycarbonyl-L-lysineesters were supplied by Fox Chemical Co. (Los Angcles, U.S.A.). N-BenzoylL-arginine amide and N-benzoyl-L-arginine ethyl ester were bought from Calbiochem. (U.S.A.) and used without further purification (they gave no detectable ninhydrin color). Ninhydrin and N-acetyl-L-tyrosinep-nitroanilide were obtained from Merck (F.R.G.). Tetranitromethane, and glycinamide were purchased from Fliika (Buchs, Switzerland). and tritiated diisopropylphosphofluol-idate from N . E . N . Cheni. (F.R.G.). Non-radioactive diisopropylphosphofluoridate, and N-tosyl-L-lysine chloromethyl ketone were purchased from Serva(Heidelberg, F.R.G.).Deuterium oxide was obtained from C.E.A. (Saclay, France) and contained 99.3",, *H,O. Methanol, isopropanol and

The enzyme was purified by affinity chromatography as described earlier [5]. from bacteria harvested at the middle of exponential growth. Estirnutioii

of Enzyme Concentrations

Trypsin solutions were prepared in 1 mM HCI containing 50 mM CaCI,, and titrated with p-nitrophenyl-p'-guanidobenzoate according to the mcthod of Chase and Schaw [13]. The protein concentration was spectrophotometrically measured at 280 nm using 14.4 [14] and 23800 as the molecular weight. On this basis, the active-site Concentration of fresh trypsin solutions was cstimated to be 57.3 7;. Protease I1 was titrated with radioactive diisopropylphosphofluoridate according to the procedure decribed by Maroux et 01. for enterokinase [15]. About 10 nmol of an enzyme preparation were dialyzed against 5 mM Tris/HCl buffer pH 7.5 containing 0.5 M KCI, and incubated with [3H]Dip-F. Knowing the molarity of the trypsin solution as measured with p-nitrophenyl-p'-guanidobenzoate, the molar specific radioactivity of the Dip-F sample could be estimated and used to calculate the active-site concentration of protease I I . Since from the analyses of the same enzyme solution the relationship between the active-site concentration and the specific activity is known. the ab-

441

M . Pacaud

solute active enzyme concentration [E,] could be obtained directly from the latter estimation. The rate assay was determined from a 1 mM solution of N benzoyl-DL-arginine p-nitroanilide in O. 1 M Tris/HCl buffer pH 8, at 410 nm and 25 "C. The protein content was measured at 278 nm using A:;: = 18.3 for protease 11. I t was found in this way that a protease preparation with a specific activity of 14300 U/mg possessed an enzymatic purity of 74%, assuming a molecular weight of 58000. On this basis a 100% active enzyme solution was calculated to have a specific activity of 19300 U/mg (average of two separate determinations).

sodium phosphate buffer pH 7. The final enzyme concentration was 0.1 1 pM. The residual activity of the inhibited enzyme was examined at a series of defined times after mixing of the enzyme and appropriate inhibitor. A 0.1-ml aliquot of the reaction mixture was withdrawn and added to 0.9 ml of 1 mM Bz-Arg-OEt in 0.1 M sodium phosphate buffer pH 7. Hydrolysis was immediately monitored at 255 nm and 25 "C. The pseudo-first-order rate constant for inactivation k(app) was calculated from the initial rates of inhibition according to Eqn (2): log ( A I I A 2 ) = k(app) ( t , - 1,) '

Radionctirity Meusurernetit

The samples of labeled enzymes (0.15 ml each) were counted in 10 ml of a dioxane-based scintillation fluid. Radioactivity was determined in an Intertechnique S2-40 liquid-scintillation spectrometer, with an efficiency of 35"" for tritium. Sirhstrute Spec(ficity Determination

The carboxymethylated B chain of porcine insulin and luteinizing hormone releasing factor were filtered through a Bio-Gel P6 column (90 x 2.4 cm) in 0.1 M acetic acid, and freeze-dried. They were digested at 37 ' C for 48 h in 2 mM ammonium bicarbonate buffer pH 8.6. diluted with water and freeze-dried. The molar ratio of enzyme to substrate was 1/50. Peptides in the digests were separated by highvoltage electrophoresis first at pH 6.5, then at pH 2.1 as described elsewhere [16]. and stained with cadmium/ ninhydrin reagent [17]. They were eluted from the paper with 0.1 M ammonia, freeze-dried, hydrolyzed in 6 M HCI (containing 0.1% thioglycolic acid) for 24 h at 110 "C in Lwcuo, and subjected to amino acid analysis. The N-terminal residues were identified as dansyl derivatives on polyamide sheets [I 81. Modificntion of' T?yrosine Residues

The general procedure of Riordan et al. [19] was used. The bacterial enzyme was dialyzed against 50 mM Tris/HCI buffer pH 7.8, and nitration was carried out by addition of a 50-fold molar excess of tetranitromethane. The reaction was allowed to proceed for 50min at room temperature, and the remainingactivity was measured on benzoyl-m-arginine p-nitroanilide. Irrewrsible Cornpetitire Inhibition

Estimation of inhibition constants for tosyl lysine chloromethyl ketone in the pH range of 6-8 were determined from Lineweaver-Burk analyses as previously described [4]. The initial rates of the irreversible inhibition were also determined at 25 'C in 0.1 M

(2)

in which A , and A , are the observed activities at times t , and t2 . The alkylation rate k2 was determined under conditions defined by Kitz and Wilson [20],from the expression

where [I] is the inhibitor concentration and Ki(app) is the dissociation constant of the enzyme-inhibitor complex. Hydrolysis o j Synthetic Substrates The kinetics of most reactions were followed spectrophotometrically at 25 (k0.2)"C. using a Beckman Acta I11 spectrophotometer equipped with a thermostated cell compartment and a recorder providing full-scale deflection for an absorbance of 0.1. Initial rates of hydrolysis of Bz-Arg-OEt and Bz-ArgNAn were measured at varied pH from substrate concentration ranging from 0.01 to 1 mM. The hydrolysis of Bz-Arg-NAn (and Ac-Tyr-NAn) was followed at 410 nm by measuring the absorption of thep-nitroanilide which is produced ( d ~ = 8 8 0 0M - ' cm-') [21]. The hydrolysis of Bz-Arg-OEt was determined at 255 nm (dt:=808 M-' cm-', [22]. Data at pH 9 and above were corrected for spontaneous hydrolysis. Under identical conditions spontaneous hydrolysis of Bz-Arg-NAn was negligible. Enzyme activities on Cbz-Lys-ONp were determined from the rate ofp-nitrophenol release at 400 nm ( A & = 16630-I cm-') [23]; values were corrected for spontaneous hydrolysis. Esterase activities on TosArg-OMe and Tvs-Lys-OMe were followed at 247 nm ( d ~ = 5 4 0 - 'cm -') [24]. Activity on Bz-Arg-NH2 was determined by measuring the concentration of ammonia released by means of the ninhydrin reaction [ 2 5 ] . Measurements of change in absorbance were converted to mol of amide hydrolyzed per min by reference to a standard curve prepared by measuring buffered solutions (25 mM phosphate buffer pH 7.8) containing various amounts of ammonium sulfate. The standard curve was found to follow the Beer-Lambert law under all

442

Protease I1 from Escherichia coli

conditions studied. Initial rates of enzymic hydrolysis were estimated as follows : 10- 50 pl of a 1 pM enzyme solution were added to 0.5 ml of substrate dissolved in 0.025 M phosphate buffer pH 7.8 (Na2HP04 KH2P04), 2 % in dimethylformamide (v/v). The reaction mixture was incubated at 25 "C and the bydrolysis was stopped by adding 1 ml of ninhydrin reagent buffered with 0.1 M sodium citrate pH 5, and placing in a boiling water-bath for 15 min. After cooling, the reaction mixture was diluted by adding 2.5 ml of a 50% ethanol solution in water (v/v), and the absorbance was measured at 570 nm. Under these conditions the molar absorption coefficient of NH: ion was found to be 1.25 x 104 M-' cm-'. At least eight different initial substrate concentrations, ranging from 0.1 to 0.8 mM, were used for each experiment. The apparent rate of ammonia released in the kinetic experiment was linear for more than 5 min. For each series of runs, parallel experiments in which the enzyme or the substrate was omitted were performed. Enzyme and substrate blanks were negligible under all conditions. The pH of each reaction solution was checked at the beginning and end of each series of kinetic runs. Throughout a series of runs, the pH did not vary by more than 0.05 unit. The steady-state parameters k,,, and K,,,(app) were determined from Lineweaver-Burk and Eadie plots. The catalytic constant k,,, is expressed by V/[E,], where V is given in mol of substrate split s-' 1-' and [E,] is the molar concentration of enzyme.

+

-

-- 0.5

0.3 -

T i -

-m

\.-

0.1

I

\

-109 [I]/ M

-

I

I

I

I

I

4.8 B

-

. I

a

4.4

4.2

7

8

PH

(3)

(4)

Kinetic data concerning pH dependence of the protease-11-catalyzed hydrolysis were analyzed according to the procedures outlined by Krupka and Laidler [26],and Alberty and Bloomfield [27]. The ratio k,,,/K,(app) depends on an ionizable basic group of pK, at low pH and an ionizable acidic group of pK, at high pH as described by Eqn (5): kca,(max)

I

5

6

It has been shown that comparison of Eqn (I), which involves an acvl-enzvme intermediate. with the usual Michaelis-Menten equation for an enzymatic process leads to Eqns ( 3 ) and (4) [6 - 71 :

-

I

I

4

Analysis of Kinetic Data

kcat

I

I

1

A

Km(app) Km(app) (max) 1 +tH +I -+ 7 Ka Kb [H 1

(5)

Fig. 1. ( A ) The influence OJ various concentrations oftosyl-L-lysine chloromethyl ketone on denree of I1 inhibition at 25 "C and _protease . p H 7; ( B ) the effect OfpH on apparent inhibition constant ofprotease I I j o r Tos-Lys-CH,Cl (0.02 m M ) at 25 "C. u is the uninhibited velocity, ui is the inhibited velocity. Activities were measured with 1 mM-Bz-Arg-OEt at 25 "C. Values of K,(app) were estimated by the method of Lineweaver and Burk

in which.k,,,(max) and K,(app) (max) are the pHindependent maximum values of k,,, and K,(app) respectively, and [H'] is the hydrogen ion concentration. RESULTS INACTIVATION STUDIES WITH IRREVERSIBLE INHIBITORS

Stoichiometry The stoichiometric inhibition of the enzyme by Dip-F was evidenced by the fact that 1 mol of 32Pwas incorporated per mol of protease I1 (see Experimental

M.Pacaud

443

Fig. 2. Inactivation of protease I I by various concentrations of Tos-Lys-CH,CI at p H 7 and 25 " C . The active enzyme concentration [E,] was 0.1 1 pM, estimated as outlined in Experimental Procedure

Procedure). With tosyl-L-lysine chloromethyl ketone, plots of log [(i3/ci)- I ] us log [I] yielded straight lines with slopes of 0.90+0.15 at pH 7 (Fig. 1A) indicating that one molecule of inhibitor interacted with each enzymatic site [28]. o/oi is the ratio of the velocities of the Bz-Arg-OEt hydrolysis without and with inhibitor, and [I] is the molar concentration of inhibitor.

1

1

30

A

p H Dependence

Competitive inhibition constant for Tos-Lys-CH2C1 were determined from pH 6 to 8. Maximum binding occurred when a single enzyme group with an apparent pK of 6.7 was ionized (Fig. 1B). Protonation of this group appears to hinder the binding of the inhibitor.

I " " " " " " '

Kinetic Analyses

To establish whether the interaction of protease TI with Dip-F and Tos-Lys-CH,C1 involves the formation of a specific enzyme-inhibitor complex, the concentration dependence of inactivation was studied according to the method of Kitz and Wilson [20]. The inhibition of the enzyme at pH 7.0 and 25 "C both by Dip-F and Tos-Lys-CH,CI at an appropriate range of inhibitor concentrations was observed to follow pseudo-first-order kinetics (Fig. 2) from which k(dpp) values were calculated. Plots of l/k(app) against l/[I] intercepted the positive y axis (Fig. 3 ) indicating saturation of the enzyme by each inhibitor with a limiting rate of inactivation k , . Estimates of kinetic constants are given in Table 1. The Ki(app) value of protease I1 for Tos-Lys-CH2C1 was about 10-fold higher than that of bovine trypsin; however, the second-order rates of inactivation were approximately the same for both enzymes (0.12 min-' cf 0.16 min-') [29].

0

0

05

10

l / [ I ](rnM-')

Fig.3. Saturation kinetics in inactivation of protease I I by ( A ) Dip-F and ( B ) 'Tos-Lys-CH2CI.Pseudo-first-order rate constants of inactivation k(app) were calculated from the initial rates of inhibition according to Eqn (2) (see Experimental Procedure)

Table 1 . Kinetic parameters for the irreversible inhibition of protease I I at 25 "C andpH 7 Experimental conditions are defined in Experimental Procedures

Tos-LYs-CHZCI Dip-F

mM

min -'

M - ' min-'

0.023 2.8

0.12 0.50

5200 178

444

Protease 11 from Escherichin coli

These observations suggest that inactivation of the bacterial enzyme could result from a functional group in the enzyme's active site, which would react covalently with either Dip-F or Tos-Lys-CH,Cl. ENZYME SPECIFICITY

Protease I1 was weakly active against the B chain of carboxymethylated insulin and long periods of digestion were required for the characterization of liberated peptides. Four ninhydrin-positive peptides were separated by high-voltage electrophoresis from a 48 h hydrolysate. The amino acid composition, N-terminal residue and relative yield of each peptide are shown in Table 2. A comparison of these data with the known amino acid sequence of the insulin B chain [30] revealed that the isolated peptides correspond to sequences 1 - 16, 1 - 22, 17- 30, and 23 -30. The enzyme appeared to cleave only two peptide bonds :

Table 2. Amino acid composition of' peptides obtained from digestion of the insulin B chain by protease I I The values in parentheses denote the theoretical number of residues of a given amino acid in the peptide Amino acid

Amount in peptide -.

1-16

1-22

-~

~~~~

17-30

23-30

1.34(1)

9.41 (1)

pmol ~

Lysine Histidine Arginine Cysteine Asparagine Threonine Glutamine/glutamic acid Serine Proline Glycine Alanine Valine Leucine Tyrosine Phenylalanine ~.

.

~

3.41 (2) 2.42 (1) 1.87 (1)

5.72 (2) 2.96 (1) 7.66(2) 3.08 (1)

2.98 (2) 1.91 (1)

9.04 (3) 3.40 (1)

1.60 ( 1 ) 7.18 (2) 1.45 (1) 3.22 (1) 2.53 (2) 7.84 (3) 4.60 (3) 12.80 (4) 1.32 (1) 3.52 (1) 1.55 (1) 3.04 (1)

1.18 (1) 1.0 (1) 1.87 (1) 1.53 ( 1 )

1.36 ( 1 ) 7.18 (1) 3.17 (2) 8.40 (1) 1.49 (1) 5.62 (1) 1.32 (1) 1.43 (1) 1.46 (1) 6.86 (1) 2.40 (2) 13.06 (2)

~~~

~~

Relative yield of peptides (%) N-terminal residue

14.8 Phe

29.4 Phe

7.90 (1)

13 Leu

~

42.8 GlY

Tyr16-Leu" and Arg2-GlyZ3in the B chain of porcine insulin (Fig.4A). However the amino acid content of the peptide GlyZ3-Ala3' suggested that the LysZ9-Ala3' bond could have been partially split. Confirmatidn was obtained by subjecting a total 48-h hydrolysate of the insulin B chain to dansylation, which revealed the presence of traces of N-terminal alanine residue in addition to the N-terminal residues identified from each isolated peptide (Table 2). These results indicate that five peptides might theoretically arise from the cleavage of the insulin B chain by protease 11. We were unable to detect one of them, the peptide Leu'' -ArgZ2. The presumed neutral charge of this peptide may have prevented its separation from the undigested B chain of insulin by electrophoresis. Protease I1 did not cleave luteinizing hormone releasing factor. The peptide obtained after hydrolysis of the hormone for 48 h at 37 "C had the same electrophoretic mobility and amino acid composition as the untreated hormone (Fig. 4B). KINETIC STUDIES OF PROTEASE-11-CATALYSED HYDROLYSIS O F SIMPLE SUBSTRATES

Dependence on Substrate Concentration

The dependence of the observed reaction velocity upon a 1000-fold range of substrate concentration (0.04 - 40 mM) exhibited a marked inhibition by excess of substrate. A conventional dependence of the initial velocity on Bz-Arg-OEt concentration, with K,(app) =0.49fO.O6mMandk,,,=200f20s-', wasobserved only at substrate concentrations lower than 1 mM (Fig. 5). Similar results were obtained with Bz-ArgNAn and Tos-Arg-OMe. Since high substrate concentrations led to anomalous kinetic behavior, the following features of the catalysis were examined at low substrate concentrations (up to 1 mM). Enzyme Reactivity towards Some Substrates

The steady-state kinetic parameters for the protease-11-catalyzed hydrolysis of several substrates were estimated in the range of optimum pH and compared with those reported for bovine trypsin. All kinetic data were determined under reaction conditions which gave initial rates in direct proportion to the substrate and

S-Tm -

S-Cm 10 15 I 20 25 30 (A) Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala 1

5

1

t (B)

t

1 5 10 < Glu-His-Trp-Ser-Tp--Gly-Leu-Arg-Pro-Gly-NHZ

Fig. 4. Summurj of resulis on the ui'tron o/ protease II uguinst tww polypeptides: ( A ) carbo.uy,,ic,th?,lc,led B choin of porcine insulin, ( B ) Iuteini2ing hormone relensing factor. Arrows indicate sites of cleavage by the enzyme

445

M.Pacaud

Table 3. Kinetic constants oj'hydrolyses o f a series oJ substrates at 25 "Cby catalyzedprotease I I and rrypsin The values for Cbz-Lys-OBzl were determined in a pH-stat in 0.1 M KCI, pH 7.2. The values for Cbz-Lys-ONp were spectrophotometrically determined in 0.05 M potassium phosphate buffer p H 7.2. Data for all other substrates were measured in 0.1 M Tris/HCl buffer pH 8. as outlined in Experimental Procedure. The hydrolysis of Ac-Tyr-OEt by protease I1 was negligible. The concentration of active enzyme [E,] was 8 t o 40 nM. the higher concentrations were used with Bz-Arg-NH, and Ac-Tyr-NAn Substrates

Protease I1

Trypsin

References __

Km (aPP)

k,,

mM

S-'

0.5 0.23 0.47 0.33 0.31

Bz- Arg-0 Et Tos-Arg-OMe Tos-Lys-OMe Cbz-Lys-ONp Cbz- L ~ s - Bzl 0 Ac-Tyr-OEt Ac-Tyr-NAn Bz-Arg-NH, Bz-Arg-NAn

80 0.6 0.29

103.

197 60 55 43 55 1.5 3.5 57

kcaI/K,(app)

M-ls-I 39.4 26.0 11.7 13.0 17.7 < 0.01 0.002 0.58 19.7

v / [ s ](min-'1

Fig. 5. Eudiu plof oJ / / i t enzymic hydrolysis oj benzoylh-urginine ethyl esler at 25 " C ,in 0.1 M TrislHCI hufferpH8. [E,] varied from 8 to 80 nM. The higher enzyme concentrations were used at substrate concentrations greater than 2 mM

the enzyme concentrations. The high K,(app) value for acetyl-L-tyrosine-p-nitroanilide and its low rate of hydrolysis rendered the determinations of k,,, and K,(app) somewhat uncertain. Kinetic constants for other substrates were estimated with an internal accuracy of about + 8 % and not worse than & 16%. As the ninhydrin method is not highly reproducible and restricts the upper limit of substrate concentration at about the value of K,,,(app), assays with benzoyl-Larginine amide were the most difficult. They were carried out at least in quadruplicate. Examination of the kinetic data for protease I1 (Table 3) indicates that, with the specific substrates listed, the values of k,,, range between 3.5 and 197 s-', and that the values of K,(app) do not differ greatly from one another. Comparatively the values of K,(app)

Km

(app)

mM 0.0026 0.015 p-nitroanilide > amide. The hydrolysis of these substrates was therefore strongly dependent on the chemical reactivity of the leaving group. The k,,, values for benzyloxycarbonyl-lysine esters cannot be explained, however, on this simple basis since the benzyl and p-nitrophenyl esters have relative rates of 1 and 45, as measured by alkaline non-enzymic hydrolysis [33]. The hydrolysis of Cbz-Lys-OBzl should be favored by a specific enzymic factor. Thus, the magnitude ofk,,, was also affected by some interaction between the enzyme and the substrate leaving group. These data indicate that some major differences exist between the catalytic site of protease I1 and trypsin. Protease I1 hydrolyzed amide and anilide substrates much better than the pancreatic enzyme, but it was about 100-fold less effective against the carboxymethylated B chain of insulin. Although the bacterial enzyme was able to cleave a Tyr-Leu bond in the latter substrate, it was weakly active against Ac-Tyr-NAn. A comparison of the k,,,/K,,,(app) ratios for Ac-TyrNAn and Ac-Tyr-OEt in Table 3 shows that protease I1 is a more specific enzyme than trypsin. Tlzermodynumic Purumeters Kinetic constants for the hydrolysis of Bz-Arg-NAn were evaluated in Tris/HCl buffer pH 8, at five different temperatures from 20 to 40 "C. The Arrhenius plot of log k,,, against 1/T gave a straight line from which an activation energy of 5.9 kcal/mol (24.7 kJ/mol) and a A S value of - 11.5 cal ( - 48.1 J) mol-' K - ' were calculated.

446

Protease I I from Escherichia coli

Table 4. The kinctic isotope effects of 'H,O on some prolease-II-catalyzed reactions Data were estimated in 0.1 M phosphate buffer (Na,HPO, + KH,PO,) at pH 7.8 and p2H 8.2. The reactions in deuterium oxide were carried out in 97.3% ,H2O with Bz-Arg-OEt, and 95.3% 'H,O with the two other substrates Substrate

H,O

Bz- Arg-OEt Bz- Arg-NH, Bz-Arg-NAn

mM 0.53 0.60 0.28

~~

S-'

184 3.4 52.6

mM 0.50 0.60 0.28

~

S-'

178 2.2 37

1.03 1.so 1.42

Efect of'Deuterium Oxide

Kinetic parameters for the enzymic hydrolysis of three substrates were measured at pH 7.8 both in ' H 2 0 and H 2 0 . Because of the broad optimum pH exhibited by protease I1 (see below), kinetic constants determined in 'H,O should be weakly affected by deuterium isotope effects on the apparent pK values of the enzyme catalytic groups. From data in Table 4, it may be seen that the replacement of water by deuterium oxide did not affect the kcatand K,(app) values for Bz-Arg-OEt ; it did, however, reduce the rate of hydrolysis of Bz-Arg-NH, and Bz-Arg-NAn by a factor of about 1.5. These variations in k,,, are small when compared to the variations expected in general base catalysis, which often demonstrated 2 - 3fold higher rates in H,O than in ' H 2 0 , as observed in the deacylation step with chymotrypsin [34] and in similar non-enzymic general base-catalyzed reactions [351. Dependence on p H

The kinetic constants k,,, and K,(app) were determined at various pH values for the protease-11catalyzed hydrolysis of Bz-Arg-OEt and Bz-Arg-NAn. As the enzyme was quickly inactivated below pH 6, the pH region studied could not be extented on the acidic side. The choice of a buffer at a pH greater than 8.5 was somewhat problematic because the k,,, values were found to be dependent on the nature of the buffer. Of the five buffers assayed, sodium borate, sodium bicarbonate, Tris/HCl,glycine/NaOH, and ammonium bicarbonate/arnmonia, the latter was the most appropriate. (The rate of Bz-Arg-OEt hydrolysis was inhibited in buffers with sodium salts, whereas it was activated in Tris or glycine buffers.)The pH dependence of the enzymic hydrolysis of Bz-Arg-NH, was not studied, as the estimates are related to the concentration of ammonia released. The important observations for the hydrolysis of Bz-Arg-OEt and Bz-Arg-NAn are given in Fig.6 and 7 respectively. For each substrate K,(app) increased at both high and low pH values, and k,,, was pH-in-

33-

2 . -8 3 2 m

E

Y

I

-

31-

' I 6

7

8

9

10

PH

Fie.6. The ~ f f ~ of c t p H on steady-stute kinetic parameters jbr the p~~~tt~use-II-catalyYen hydrolysis of Rz-Arg-OEt. The dashed line is a theoretical curve calculated from the pK values of k,,,/K,(app) gi\cn in Table 5. Experimental data are average values from the mean of three separate experiments. [E,] varied from 8 to 40 mM. The rate of reaction was proportional to enzyme concentration over t h i 4 range. The buffers used are given in Experimental Procedure

dependent over a large range of pH. The apparent pK values determined from the experimental curves (Fig. 6 and 7) by the method of Dixon [36] are summarized in Table 5. Since ionization constants of the substrates lie outside the pH range of the experiments, they need not be considered here. The shape of the k,,,/K,(app)

M. Pacaud

447

effect does probably exist because the apparent pK, and pK, values for Bz-Arg-OEt and Bz-Arg-NAn differ by about 0.8 pH unit. The shape of the k,,, vs pH curves of the ester and anilide was essentially the same ; the catalysis of these two substrates appears to follow the same pathway. The dashed lines in Fig.6 and 7 represent the theoretical curves derived from Eqn (5). The values of the ratio k,,,/K,(app) (max) used for these calculations were 372 x lo3 M-' s-l for Bz-Arg-OEt and 195 x lo3 M-' s-' for Bz-Arg-NAn. The experimental data fit the theoretical curves very well on the acidic side, but they diverge on the alkaline side mainly for Bz-ArgOEt. The reason for this deviation was not explored.

13t

i

NUCLEOPHILIC COMPETITION

I

6

7

t

1

8 PH

1

1

1

9

1

10

Fig.1. The Kflec.1 UJ pH on sleudy-state kineric parameters for [he prorecise-11-calalyzed hydrolysis of Bi-Arg-NAn. The dashed line is a theoretical curve calculated from Eqn (5). Experimental data are average values from the mean of three separate experiments. The reaction mixture contained 2% dimethylformamide. [E,] varied from 3.5 to 17.5 nM. The rate of reaction was proportional to enzyme concentration over this range

Table 5 . Apparent pK values for protonic ionizations afli,cting the protease-11-catalyzed hydrolysis of Bz-Arg-OEt and Bz-Arg-NAn Substrate

Kinetic constant used for calculation

pK,

PK,

Bz-Arg-OEt

Km(aPP) km kca,/Km(aPP)

6.7 6.7 6.7

8.1 28.7

vs pH curves suggests the participation of two ionizable groups in the catalytic hydrolysis of both substrates. Since the pK(app) values of these groups represent the ionization of the enzyme they should be identical for all substrates [37], provided that there is no pH-dependent interaction of positive substrate with non-catalytic groups on the enzyme. Such an

The effects of some organic solvents and glycinamide on reactions catalyzed by protease I1 were studied at 25 "C and pH 7.8. Kinetic parameters for the hydrolysis of Bz-Arg-OEt were estimated from the initial release of the P, product (Bz-Arg), while for the hydrolysis of Bz-Arg-NH, and Bz-Arg-NAn they were estimated from the initial release of the PI product (NH, or p-nitroanaline), as described in Experimental Procedure. A solvent concentration range between 0 and 10% (w/w) was generally used because higher concentrations inactivated the enzyme. The effects of methanol and dioxane were investigated together in order to dissociate an eventual participation of the nucleophilic reagent in the enzymic process from secondary effects resulting from a lowering of the dielectric constant of the reaction medium [38,39]. In the cleavage of Bz-Arg-OEt and Bz-Arg-NH, both k,,, and K,(app) increased with the increasing concentration of each solvent, with the exception that dioxane had a negligible effect on k,,, in the amide cleavage. The dependence of these kinetic parameters on the dielectric constant of the medium is illustrated in Fig. 8 and 9. The k,,, or K,(app) values for the ethyl ester, which were determined in either methanol, isopropanol, or dioxane, fall on the same straight line (Fig.8). Thus the effect of these solvents on the hydrolysis of Bz-Arg-OEt is due to their effect on the dielectric strength of the medium. The K,(app) values, but not the k,,, values, for the amide bear the same relation whether methanol or dioxane was present in the reaction mixture (Fig. 9) (the effect of isopropanol was not tested for this substrate). The plot in Fig. 10 shows the influence of the methanol concentration on k,,,. A linear relationship was observed in the range of alcohol concentrations used. Methanol therefore appears to exert a nucleophilic effect only through an increase in the rate constant of the Bz-Arg-NH, hydrolysis. The k,,, value for Bz-Arg-OEt was unaffected in the presence of 0.1 M glycinamide but the K,(app)

448

Protease I I from Escherichiu coli

-32-

. I

Y 2 8 a E 3 3 01 -

I

1.26

_____(_\: A

29-

I

128

130

I

I

132 134 10'. I / D

I

136

I

138

140

Fig. 8. The dependence of log k,,, and log K, on the reciprocal ofthe dielectric constunt,fiw the proreu.Fe-Il-catalyzed hydrolysis of 'Bz-ArgOEt. Dioxane(0); isopropanol (A); methanol (0).Kineticconstants were estimated in 0.1 M phosphate buffer (Na,HPO,+KH,PO,) at pH 7.8 and 25 C. The solvent/water mixtures were prepared as outlined in Experimental Procedure

09

05

126

128

130

132

134

1.36

138

140

10'. 1/D

Fig.9. The dependence nf log k,,, und log K, on the rrciprocul nf the dielectric constunt,for the proteuse-Il-cata1.vzed hydrolysis of Bz-Arg-

N H , . Dioxane (a); methanol (0). Experimental conditions are given in Fig. 8

value increased somewhat (0.67 cf. 0.5 mM). The k,,, values for Bz-Arg-NAn were 83 s - l and 57 s-' and the corresponding K,(app) values were 0.57 and 0.29 mM in the presence and absence of 0.1 M glycinamide, respectively. Although glycinamide binds to the active site of protease I1 its effect on k,,, is in agreement with that of methanol. Both reagents accelerate the liberation of the P, product from the amide cleavage and d o not change the liberation of the P2 product from the ester cleavage.

Methaml ( M )

Fig. 10. The dependence ($k,,,,,, on rnethunol concentrution for the enzymic hydrolysis of Bz-Arg-NH, , k,,,(obs)/k,,,(H,O) is the ratio

of rate constants estimated in the presence and absence of methanol

DISCUSSION SpeciJicity and Kinetic Properties Studies of the specificity of protease I1 towards polypeptides revealed a very limited proteolytic activity. A 48-h digestion of the carboxymethylated B chain of insulin led to the hydrolysis of two peptide bonds formed by the carboxyl group of arginine-22 and tyrosine-16. The major peptide obtained in about a 40% yield was composed of residues 23 - 30, indicating that the main point ofcleavage occurred at the Arg-Gly bond of the insulin B chain. Luteinizing hormone releasing factor was not split at all under the same conditions. Like trypsin, protease I1 was unable to cleave Arg-Pro bond [40] (see Fig. 1). The cleavage of the Tyr-Leu bond in the B chain of insulin was not expected, since the bacterial enzyme does not hydrolyse Ac-Tyr-OEt and cleaves Ac-Tyr-NAn non-specifically . Furthermore two other tyrosyl bonds present in the two polypeptides tested were completely resistant to hydrolysis. There is no apparent analogy between the amino acid residues present on either side of the two susceptible bonds in the insulin B chain ; nevertheless, trypsin can also split some bonds involving the carboxyl of an aromatic residue on prolonged periods of incubation [41]. The lack of significant hydrolysis of the lysyl bond adjacent to the carboxyl group of the terminal alanine residue suggests that protease I1 might well have a substrate specificity comparable to that of thrombin [42]. The competitive inhibition of the hydrolysis of various small substrates by L-arginine and not by L-lysine [4,5] is also consistent with a preferential binding of the arginine moiety to the active site of the enzyme. In spite of its low activity towards peptide bonds, protease I1 readily hydrolyses typical synthetic substrates of trypsin, but the enzymes were considerably different by kinetic parameters. The most striking feature of the data summarized in Table 3 is the lack of large variations in the K,(app) values of the bacterial enzyme among substrates of different susceptibility. These results suggest that the K,(app) for these

M . Pacaud

substrates approximates K, , the dissociation constant of the enzyme-substrate complex. Such an interpretation is also consistent with the fact that in the hydrolysis of N-benzoyl-L-arginine derivatives, k,,, and K,(app) do not show parallel changes with modifications of pH. The k,,, values for Cbz-Lys-ONp and Cbz-Lys-OBzl are very different from the rates of the alkaline hydrolysis of these derivatives. This implies a specific interaction of the substrate leaving group with the active site of the enzyme. Moreover, it is noteworthy that protease I1 hydrolyses a-N-subtituted arginine esters and N-benzoyl-L-arginine-p-nitroanilide with a comparable catalytic efficiency. A comparison of the rate constants for Bz-Arg-NH, and Bz-ArgNAn suggests that the interaction of the enzyme with the hydrophobic part of the substrate might favor the cleavage of the amide bond. Trypsin and protease 11, on the other hand, exhibit a different kinetic behavior at high substrate concentration. While the pancreatic enzyme is activated by an excess of substrate [43,44], the bacterial enzyme is strongly inhibited in the presence of both esters and amides above the concentration of 1 mM. The mechanism of substrate inhibition was not studied in detail, but inhibition by the acid part of substrates (P, product) appears unlikely because the Ki(app)value for N-acyl-L-arginine is largely below the K,,,(app) for all substrates [5]. If a substrate is attracted to the enzyme in more than one possible arrangement as is supposed, then two or more molecules of substrate might be adsorbed on the active site [451. The hydrolysis of p-nitrophenyl-p’-guanidobenzoate, the active-site titrant for trypsin, followed slow first-order kinetics but no ‘burst’ release of p-nitrophenol was observed, further indicating significant differences between the catalytic site of trypsin and protease 11. Nature of the Amino Acid Residues Involved in the Active Site Modification studies have shown that the bacterial enzyme reacts stoichiometrically with both diisopropylphosphofluoridate and N-tosyl-L-lysine chloromethyl ketone with a concomitant loss of activity. On the basis of kinetic data it may be inferred that each compound binds to the active site to form a stable complex which is catalytically inactive. Dip-F inactivates bovine trypsin by binding to the hydroxyl of serine-183 [46], while the site of substitution of TosLys-CH,CI has been identified as the N-3 ofhistidine-46 [47]. By analogy with the pancreatic enzyme, it is then assumed that the integrity of seryl and histidyl residues in the active site of the bacterial enzyme are essential for enzyme activity. Because of the low concentration of enzyme in Escherichia coli cells, it does not seem possible to isolate the modified peptides and to identify

449

the specific residues, the modification of which is responsible for loss of activity. The involvement of a histidyl residue in the active site is supported by the effect of pH on the binding of Tos-Lys-CH,CI, which indicates ionization of a basic group with an apparent pK of 6.7. The variations with pH of K,(app) and k,,,/K,(app) for the hydrolysis of two related substrates, Bz-ArgOEt and Bz-Arg-NAn (illustrated in Fig.6 and 7 respectively) suggest the presence of two ionizable groups in both the enzyme-substrate complex and in the free enzyme. One has a pK(app) around 7, the other has an average value of 9-9.5. Theoretical curves that fit the data are also consistent with the presence of two enzymic groups, one acting as a base and the other as an acid. The apparent pK value in the range of pH 6.7-7.5 is in agreement with the pK, value determined from the enzyme inactivation by Tos-Lys-CH,CI. Thus, it seems reasonable to assume that the same histidyl residue is responsible for these observations. If such a group is involved in the catalysis it is functional only in the protonated state, i.e. it acts as a proton donor during catalysis. In a-chymotrypsin [48] and Myxobacter a-lytic protease [49] the pK of about 7 should be in fact assigned to aspartate-102 in the active-site triad Asp-Ser-Gly. The assignment of the pKin the range of pH 8.7-9.5 is not obvious. This pK range is characteristic of both a and E ammonium groups, thiol or phenol hydroxyl group. Since sulfhydryl reagents [4] and tetranitromethane do not affect the enzyme activity, the involvement of a cysteinyl or tyrosyl residue appears unlikely. The pH dependence of the enzyme inactivation in 2.5 M urea exhibited a sharp decrease between pH 8 and 10 (unpublished data) suggesting some alteration in the folding of the enzyme in this pH region. Perturbations in the k,,,/K,,,(app) curves on the alkaline side could reflect, at least in part, the same phenomenom. Moreover kcatwas found to vary with the nature of the alkaline buffer used. Added nucleophiles also affect the k,,, value of amide substrates, and the increase in the rate constant above pH 9.4 for Bz-Arg-NAn might arise from some interaction with a nucleophilic buffer component like ammonia. The NH: species should be the reactive one since the effect increases with increasing pH. If K , is related to K,, it may be inferred that the ionizing group with a pK(app) of 8.7 for Bz-Arg-OEt and 9.6 for Bz-Arg-NAn affects the binding of substrates but not their rate of hydrolysis. The hydrolysis of synthetic substrates by a-chymotrypsin is dependent on a group with a pK of 8.5, which has been associated with its NH,-terminal isoleucine residue [50]. The state of ionization of this group controls the active conformation and the substrate binding ability of the enzyme [51,52]. For both trypsin and a-chymotrypsin, whose tertiary structures have been elucidated by X-ray diffraction, the same a-NH,

Protease I1 from Escherichia coli

450

group was shown to form a salt bridge with Asp-194 [53,54]. In the case of bovine trypsin the pK of 10, which controls the hydrolysis of synthetic substrates [55,56], is also assigned to such an interaction. From these considerations it is tempting t o speculate that the group having a pK around 9 in the Escherichia coli enzyme might be required for the maintenance of an appropriate conformation of the active site, rather than being directly involved in thecatalytic mechanism. Scheme A E+S-

k3(H20)

K,

E+P,

k E S L ES'

+ PI

E+P,

A nucleophilic attack of ester substrates by the enzyme might account for the absence of a nucleophilic effect on the rate of Bz-Arg-OEt hydrolysis [60], but the results obtained for amide substrates are inconsistent with both Schemes A and B. One possible explanation of these findings could be that both reagents bind to the enzyme and increase the rate of formation of the intermediate (k2).The data in methanol were corrected for the dielectric constant increment, but nucleophiles are liable to exert other secondary effects. Although the experiments were performed at low nucleophile concentrations, effects on the enzyme conformation or on the stability of transition states and intermediates cannot be excluded.

Partitioning of the Intermediate

Scheme A gives the course of a reaction catalyzed by pancreatic serine hydrolases in which the solvent contains water and an added nucleophile (ES' is the acyl-enzyme). I f the model is applicable to the present system, the effects of added nucleophiles may be easily interpreted with the help of the analytical treatment developed by Seydoux and Yon [57] from the equations etablished by Bender er al. [58]. The invariability of K,(app) for the protease-11-catalyzed hydrolysis of both Bz-Arg-OEt and Bz-Arg-NH, in the presence of methanol provides a further argument that K, is an equilibrium binding constant. With substrates for which K , approximates K, it is expected that kc,,(,, (for the P, product) will decrease linearly with increasing nucleophile concentration, while kc,t(l) (for the PI product) will remain constant. The increase in k,,,cl, for both Bz-Arg-NH, and Bz-Arg-NAn in the presence of methanol and glycinamide respectively, and the absence of change in k,,,,,)for Bz-Arg-OEt in the presence of both reagents (Fig.8- 10) are, therefore, incompatible with the predictions formulated from the acyl-enzyme mechanism. Another possibility could be that the reactions catalyzed by protease I1 proceed through a tetrahedral intermediate (ES*) as represented in Scheme B [59]. Scheme B KS k E+S ES 2 ES*

E+PP,+P2 k4(N)

E + PI + P3

Such a mechanism has been proposed for some chymotrypsin-catalyzed reactions [59,60]. The following equations :

and

-d[P31- ES* k,(N)

dt are common to Schemes A and 9, nevertheless the magnitude of k,(N) can be different in each scheme.

Mechanistic Proposals If the breakdown of an acyl-enzyme intermediate is the rate-limiting step, it may be expected that under suitable conditions a burst release of the acyl product should be observed during the presteady-state reaction. Efforts to detect such a burst were unsuccessful with p-nitrophenyl-p'-guanidobenzoate.The rate constant k,,, for amide and ester substrates depends significantly on the nature of the leaving group; these results are clearly incompatible with a mechanism in which a common acyl-enzyme intermediate is formed. Since K , represents the thermodynamic dissociation constant oftheenzyme-substratecomplex, K,(i.e.k, Qk-,). the rate-limiting step is that which immediately follows the formation of the Michaelis complex. Consequently no kinetically significant intermediate accumulates in the pi-ocess. The differences between the rates of non-enzymic base-catalyzed hydrolysis of alkyl esters and corresponding amides is commonly 1@-10". By contrast, the ester :amide-hydrolysis rate ratios for nonenzymic acid-catalyzed reactions are commonly less than 10 [61]. The relative rates of the protease-11catalyzed hydrolysis of Bz-Arg-OEt and Bz-Arg-NH, , kcal(ester)/k,,,(amide)= 60, indicate that electrophilic and nucleophilic components seem to play a role in the catalytic mechanism. On the other hand, the low deuterium isotope effect on k,,, for esters and amides (Table 4) suggests that the nucleophilic component might be involved in the rate-limiting step. Conclusion

The above data lead to the assumption that the hydrolytic reactions catalyzed by protease I1 proceed through a tetrahedral intermediate the formation of which is the rate-limiting step. The elucidation of the catalytic mechanism, however, requires much further work. In the hydrolytic reactions catalyzed by a-chymotrypsin [62], elastase and a-lytic protease [63], it has been shown that a tetrahedral intermediate be-

M. Pacaud

45 1

tween the Michaelis complex and the acyl-enzyme does exist but does not accumulate to an observable degree (less than 49.i). In contrast to other serine proteases whose mechanisms have been elucidated, protease I1 is an endocellular protease. Whether or not thecatalytic features of this enzyme may be related to its biological function is unknown. 1 a m indebted to Dr G . Orsini for his help in the pH-stat measurements. I a m very grateful t o Dr G . N . Cohen. in whose laboratory the specificity studies were carried out, and also to G . LeBras and M. Gouyvenoux for the performance of the amino acid analyses. I would like to extend my gratitude t o Dr F. Seydoux and J. J. Bechet for helpful discussions and critical reading of the manuscript. Finally. I would also like to thank Dr J. Uriel for support and careful reading of the manuscript.

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M. Pacaud. Centre dc Biologie Moleculaire du C.N.R.S.. 31 Chemin Joseph-Aiguier, F-I3274 Marseille-Cedex-2. France