Eur. J. Biochem. 196, 385-393 (1991) 0FEBS 1991

001429569100155R

Functional residues at the active site of aminopeptidase N Armelle HELENE, Ann BEAUMONT and Bernard P. ROQUES Departement de Chimie Organique, Unit6 266 de I’Institut National de la Santt et de la Recherche Medicale, Unite AssociCe 498 du Centre National de la Recherche Scientifique, Unite de Formation et Recherche des Sciences Pharmaceutiques et Biologiques, Paris, France (Received October 10,1990) - EJB 90 1214

Sequence analysis of aminopeptidase N has shown that this zinc exopeptidase contains a consensus sequence

(Val-Xaa-Xaa-His-Glu-Xaa-Xaa-His), generally found at the active site of zinc endopeptidases [Jongeneel, C. V., Bouvier, J. and Bairoch, A. (1989) FEBS Lett. 242,211 -2141. This suggests that the active site of aminopeptidase N may be closer to that of a classical zinc endopeptidase, such as thermolysin, than to that of an exopeptidase, such as carboxypeptidase A, which does not contain the above sequence. However, the nature of the other amino acids involved in the enzymatic activity of the eukaryotic aminopeptidase N remains unknown. Chemical modifying agents have now been used to characterize the active site of aminopeptidase N further. The location of the modified residues was also determined by comparing the protection given by three competitive inhibitors which interact with different subsites of the active site. Aminopeptidase N was rapidly inactivated by 2,3-butanedione and diethylpyrocarbonate and partially inactivated by N-acetylimidazole, diazoacetamide and a soluble carbodiimide, suggesting the presence of functional arginyl, histidyl, tyrosyl and aspartyl/glutamyl residues. In each case the reaction kinetics showed that the inactivation could be correlated with modification of a single residue. The protection experiments indicated that the residues are at the active site of the enzyme and that the arginine and tyrosine are probably located in the S;-Si subsites, histidine in the S1 subsite and the acidic residue near the zinc binding site and the S; subsite. Steady-state kinetics showed that the arginine, histidine and acidic residues are involved in substrate binding, while the tyrosine may play a role in the catalytic process. All these data support an endopeptidase-like structure for the active site of aminopeptidase N. Aminopeptidase N is a zinc-containing proteolytic ectoenzyme which removes the N-terminal amino acid of protein and peptide substrates. It is a stalked integral membrane protease, mainly located in the small intestinal and kidney brush borders but also found in brain, lung, liver and primary cultures of fibroblasts [l -61 and shown to be identical to the myeloid leukemia marker CD13 [7]. In the brain, aminopeptidase N has been found to be involved in the degradation of neuropeptides, particularly the endogenous opioid peptides, enkephalins [8, 91, in association with neutral endopeptidase [lo], another zinc metallopeptidase [ll]. Mixed inhibitors of both aminopeptidase N and neutral endopeptidase, such as kelatorphan 1121, have been shown to completely block the in vivo metabolism of enkephalins [13] leading to strong spinal [14] and supraspinal analgesic effects [15]. Mixed inhibitors, as well as selective aminopeptidase N inhibi-

tors, have been designed by using a simplified model of a zinc metallopeptidase active site 1161, since little is known about the structure and the molecular mechanism of action of this enzyme. Structure/activity studies using different peptide substrates, bestatin and analogues, thiol or bidentate-containing inhibitors [17 - 191 have shown that this enzyme preferentially removes amino acids with hydrophobic side chains and interacts strongly with compounds possessing aromatic residues in the PIand P i positions. Recently, aminopeptidase N from human 1201, rat [21, 221 and pig [23] have been cloned and sequenced and their respective amino acid sequences show large similarities. This contrasts with the lack of significant sequence similarities with other zinc metallopeptidases, except for the consensus sequence Val-Xaa-Xaa-His-Glu-Xaa-Xaa-His, which contains essential amino acids present in the catalytic site of the bacterial endopeptidase thermolysin [24] and is also found Correspondence to B. P. Roques, Laboratoire de Chimie Or- in the superfamily of zinc endopeptidases such as neutral ganique, INSERM U 266,Centre National de la Reserche Scientifique endopeptidase, angiotensin-converting enzyme, and members UA 498,Faculte de Pharmacie, 4 avenue de I’Observatoire, F-75006 of the mammalian collagenase family [25]. Interestingly, unlike Paris, France aminopeptidase N, the consensus sequence Val-Xaa-Xaa-HisAbbreviations. EDC, l-ethyl-3(3-dimethylaminopropyl)carbo- Glu-Xaa-Xaa-His is absent in the zinc carboxypeptidases A, diimide; bestatin, (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-~B and E 1251, suggesting that, in some aspects, the activeleucine; inhibitor 1,3-amino-4-phenyl-N-hydroxybutanamide; in- site structure of aminopeptidase N may be closer to a zinc hibitor 2, ~3-(hydroxyaminocarbonyl)-2-benzyl-l-oxopropyl]-2endopeptidase model than to that of a classical zinc exoaminocyciopentane carboxylic acid. Enzymes. Aminopeptidase N (EC 3.4.11.2);neutral endopep- peptidase. In order to elucidate the molecular organization of tidase (enkephalinase) (EC 3.4.24.11); thermolysin (EC3.4.24.4); carboxypeptidase A (EC 3.4.17.1);angiotensin-converting enzyme the active site of aminopeptidase N, we have used chemical (EC 3.4.15.1). modifications by selective reagents. This approach was re-

386 inforced by protection experiments with three competitive inhibitors, which bind to different subsites of the enzyme. The results of these studies provide evidence for the presence of tyrosyl, arginyl and carboxyl groups in the S1-S; subsites and a histidine in the S1 subsite involved in binding and/or in the catalytic activity of aminopeptidase N, demonstrating an endopeptidase-like organization of its active site.

Absorption spectra

MATERIALS AND METHODS

Kinetics of inactivation

Aminopeptidase N (2 mg) was incubated either with 1 0 m M diethylpyrocarbonate or with 20 or 30 mM Nacetylimidazole in the appropriate buffers for 1 h in a total volume of 1 ml and excess reagent removed as described above. Absorption spectra of the modified and native enzymes were then performed on a Beckman model 35 double-beam spectrophotometer in 3.0-ml spectrophotometer cuvettes.

Materials

Different dilutions of the reagents were freshly prepared Diethylpyrocarbonate, N-acetylimidazole, l-ethyl-3(3- and preincubated at 25°C for 10 min. Enzyme was added to a dimethylaminopropy1)carbodiimide (EDC) and hydroxyl- final concentration of 70 nM to initiate the reaction. Aliquots amine were purchased from Sigma, and 2,3-butanedione from were removed at regular intervals and diluted in an excess of Janssen. Diazoacetamide was prepared according to the the Tris buffer to stop the reaction. Remaining enzymatic method of Grossberg and Pressman [26]. [tyro~yl’-3,5-~H, activity was measured as described above. Kinetics of inactiLeu’IEnkephalin ([‘H’, Leu’lenkephalin) (50 Ci/mmol) was vation were expressed by the exponential equation In [A/ obtained from Amershani and [Leu’]enkephalin from A,] = - kobst,where A is the enzyme activity in the presence Bachem. Aminopeptidase N (amino acid arylamidase) from and A, in the absence of the reagent. The pseudo-first-order hog kidney was purchased from Boehringer Mannheim as a rate constant for inactivation (kobs)and second-order rate constant (k,) were related by the equation: suspension in 3.2 M ammonium sulfate, 50 mM Tris buffer.

(1 1 where [R] is the chemical reagent concentration and n is the minimal number of R molecules needed to inactivate a single molecule of active enzyme. The kinetic order of the modification reaction, n, was obtained from the logarithm form of the Eq. (1): kobs

Inhibitors Bestatin ( K , = 0.5 pM) was purchased from Roger Bellon Laboratories, France. Inhibitor 1, i. e. 3-amino-4-phenyl-Nhydroxybutanamide, which h&s a K, of 29 pM for aminopeptidase N. was a generous gift from Professor M. C. Fournie-Zaluski. Inhibitor 2, i. e. N[3-(hydroxyaminocarbony1)-2-benzyl-1-oxopropyl]2-aminocyclopentane carboxylic acid, was synthesized in our laboratory as previously described [I91 and has a K, of 0.16 pM.

Treatnwnt of’amiflopept iduse N 1 vith group-specqic reagents Amino acid modifications were carried out in the following buffers: 50 mM Hepes pH 7.4 at 25°C for N-acetylimidazole, 0.1 M phosphate pH 7.2 at 25°C for diethylpyrocarbonate, 50 mM borate pH 8.2 at 30 ’C for 2,3-butanedione, 0.1 M phosphate pH 6 at 25 C for EDC and H 2 0 at 25°C for diazoacetamide. 1 pg enzyme was incubated with the different reagents in a total volume of 100 pl (70 nM aminopeptidase N final). Reactions were stopped by separating enzyme from excess reagent on a calibrated Sephadex G-25 column (Pharmacia, PDIO) equilibrated either in 50 mM Tris/HCl pH 7.4 at 4°C or 0.1 M phosphate pH 6 at 4°C when EDC was used as the modifying agent. Aminopeptidase N, incubated with appropriate buffer in the absence of reagents and treated as described above, was taken as a control. Deterin inat ion of’ enz!.nie ai,tivity Residual activity was measured using an aliquot of the eluted enzyme and [ 3 H I , Leus]-enkephalin as substrate. Modified and control enzymes were incubated for 15 min at 25°C in 50 mM TrisjHC1 pH 7.4 with 10 nM [3H1,Leus]enkephalin, and the initial rate of [3H]tyrosine formation was measured as previously described by separation of the tritiated metabolites formed on polystyrene beads [27]. IC50values were calculated by preincubating the enzyme for 10 min with increasing concentrations of inhibitor before addition of the substrate.

= k,“”

ln(kobs)= n . ln[R]

+ ln(kl)

Inhibitor protection experiments Protection from inactivation by specific competitive inhibitors was carried out by preincubating the enzyme for 15 min at 25°C with 2 mM inhibitor before adding the amino acid reagent. The reaction was stopped by separating the enzyme from inhibitor and excess reagent over a Sephadex G-25 column as described above. Efiect of modfication on steady-state kinetics

The K, and V,,, values for [Leu’lenkephalin were determined by the method of Eadie and Hofstee with substrate concentrations ranging over 6.25 - X00 pM and I0 nM [’HI, Leu5]enkephalin included as a tracer. In these experiments, as in the inhibitor protection experiments, diethylpyrocarbonate and butanedione were used at 10 mM for 1 h, N-acetylimidazole at 20 mM for 1 h and diazoacetamide at 500 mM for 4 h. Protein determination Protein concentrations were determined by the method of Bradford with crystalline bovine serum albumin as standard

PSI. RESULTS Selection of inhibitors In order to investigate the active-site localizations of the amino acids modified by the chemical reagents, the aminopeptidase N inhibitors 1 and 2 were used (Fig. 1). Inhibitor 1 was designed to interact with the s, subsite and

387

R' I

H,h-CH-C

B

YH2 O II H,~-CH-CH~-C-NH

Y

H

H

I

I

Zn2'

A

X

0

II

-NH-CH-C-NH-CH-C-NHR

e' I

-

I

(A- -

~

I

0

II

I

OH I

fi

I 1.2

HO 0

I I1

C

HN-C-CH,-CH-

C-NH

c-o-

0

D

I

I

I

I

H&-CH-CH-C-NH-CH-

hH2 0

I

.

\A I

10

I

I

20

30

I

40

50

TIME min -.--

kHz OH 0

!

IIU R

.

-2~n .

Ib

/

' 1I

/ 'I

I

I1

C-O-

Fig. 1. Schematic models for the binding of putative substrate ( A ) , inhihitor 1 ( B ) , inhibitor 2 ( C ) and bestatin ( D ) to the uctive site of uminopeptiduse N

inhibitor 2 with the S;-S; subsites. The differential protection provided by these two inhibitors against chemical modification gives an indication of the localization of the modified residues in the different subsites of the active site. As shown in Fig. 1, both compounds have a hydroxamate group located at the position of the scissile peptide bond of normal substrates, and hydrophobic side chains. By analogy with thermolysin, for which the structure of the hydroxamate inhibitor complex is known [29], it is assumed that the hydroxamate moiety binds to the active-site zinc atom. However, owing to the strict exopeptidase action of aminopeptidase N, the phenylalanyl and aminocyclopentane carboxylic residues of 2 bind to the S; and S; subsites, respectively, to allow the coordination of the hydroxamate. In compound 1,the localization of the phenyl moiety in the S1 subsite is directed by the well-known strong stabilization ensured by the amino group placed in the same position as that of a N-terminal group of a substrate [17]. Bestatin, a well studied inhibitor of aminopeptidase N [17], was taken as a control for the protection experiments. The 2-hydroxy-3-amino-4-phenyl butanoic acid portion of bestatin is thought to bind to the S1 subsite, owing to the aminopeptidase nature of the enzyme, and the amide carbonyl and 2-hydroxy substituent are the proposed zinc ligands, as shown in Fig. 1. Inhibition of uminopeptiduse N by butanedione

All experiments were performed in the dark to prevent photochemical destruction of histidines and tryptophans by 2,3-butanedione [30]. Butanedione at 10 mM decreased enzyme activity to about 60% of the control in 50 mM Hepes pH 8.2 and to less than 30% in 50 mM borate pH 8.2 in 1 h. The enhancement of inhibition by borate buffer is an indication of a butanedione-induced arginine modification, since the formation of a borate complex stabilizes the unstable 4,5-dimethyl-4,5-dihydroxy-2-imidazoline intermediate issuing from the reaction between one molecule of butanedione

0.02

O !/ 0

I

I

10

20

[ BUTANEDIONE]

mM

Fig. 2. Inactivation ofarninopeptiduse N by butanedione. (A) Effect of various concentrations of butanedione on the rate of inactivation of aminopeptidase N. The enzyme (1 pg) in 50 mM borate pH 8.2 was treated in the dark with: 0 ( O ) ,3.5 mM (0),5 mM ( x ), 9 mM (m), 15 mM ( A ) and 20 mM (A)butanedione at 30°C. At time intervals, aliquots were removed for measurements of the residual enzyme activity. The first-order rate constants kobs were determined from the slopes of the straight lines. (B) Dependence of /cobs on [butanedione]. Kobsfor inactivation was linearly dependent on the concentration of butanedione and the second-order rate constant kl was calculated from the slope of the line. (C) In kobsplotted against In [butanedione]. The slope of the line gives the reaction order with respect to butanedione

and the guanidinium group of one arginine [31, 321. The time course of aminopeptidase N inhibition by butanedione in borate buffer is shown in Fig. 2. Rates of inactivation appeared to obey pseudo-first-order kinetics (Fig. 2A). A plot of the first-order rate constant kobs as a function of inhibitor concentration was linear, giving the second-order rate constant k l = 3.4 M-' min-' at 30°C and could be extrapolated to the origin, indicating irreversible modification (Fig. 2 B). The l n / h plot kobsversus reagent concentration had a slope of 0.92 (Fig. 2 C) suggesting that one molecule of butanedione is needed to inactivate one molecule of enzyme. The known specificity of butanedione as an arginine reagent [33], the enhancement of inhibition by borate buffer and the kinetics

388 Table 1. Protection by competitive inhibitors uguinst group reagent inactivation of arninopeptidase N All incubations wcrc carried out at 25"C, except for butanedione (30°C). Aminopeptidase N was preincubated with 2 mM inhibitor for 15 rnin and the reagents were then added. Reactions were stopped by gel filtration on a Sephadex G-25 column and aliquots of the eluted enzyme were used for measuring enzymatic activity. Aminopeptidase N preincubated with the buffer only and treated as described above was taken as a control. Data are the mean values of three independent experiments performed in duplicate Reagent

Concentration

Incubation time

Protection by 2 mM bestatin

Butanedione Dieth ylpyrocarbonate N-Acetylimida7ole Diazoacetamide

mM

min

%

I0 10 20 500

60 60 60 320

40 f 5 100 f 2 100 f 2 100 f 2

inhibitor

1

2 f 1 100 f 0 53.0 f 17 100 f 2

inhibitor

2

56.0 f I1 .O 13.8 & 6 100 f 2 100 1

*

Table 2. Kinetic pamrneters ?f[Leu5]enkephalindegradation by aminopeptidase N before and after modification by specific amino-acid reagents Butanedione and diethylpyrocarbonate were added to a final concentration of 10 mM; N-acetylimidazole was used at 20 mM and diazoacetamide at 500 mM. Incubations were carried out at 25°C (30°C for butanedione) in the indicated buffers. Reactions were stopped by gel filtration after 1 h (except for diazoacetamide: 4 h). Aminopeptidase N incubated with the buffer only was taken as a control. The data are the mean values of four experiments performed in duplicate Reagent

Buffer

Butanedione Diethylpyrocarbonatc N-Acetylimida7ole Diazoacrtamidc

borate phosphate Hepes H2O

Control aminopeptidase N

Modified aminopeptidase N

PM

pmol , min-' . ng-

142.2 & 28 65.5 f 13.1 86.3 rl: 26 87.6 20.9

0.357 f 0.091 0.388 f 0.078 0.376 rl: 0.081 0.1 54 & 0.04

of inhibition suggest that specific modification of a single essential arginine leads to loss of enzymatic activity. Bestatin and inhibitor 2 at a concentration of 2 mM partially protected (40 - 50%) aminopeptidase N against butanedione action whilst 2 mM inhibitor 1 had no effect (Table 1). These results indicate that the modified residue is at the active site and probably at the S;-Si subsite level. After a 1-h incubation of aminopeptidase N with 10mM butanedione, the K, of [Leu5]enkephalin for the enzyme was found to have increased threefold in comparison to the control enzyme, with no change in V,,, (Table 2), suggesting a predominant role of the arginine in substrate binding. Inactivation of aminopeptidme N b j diethylpyrocarbonate Inactivation of aminopeptidase N by diethylpyrocarbonate in 0.1 M phosphate pH 7.2 also exhibited pseudo-firstorder kinetics (Fig. 3). Despite the presence of the reagent in a large molar excess over the enzyme, a semi-log plot of residual enzyme activity versus time did not yield a straight line and the rate of inactivation decreased with time (Fig. 3A). This may be due to the instability of diethylpyrocarbonate since this reagent is readily hydrolyzed in aqueous solution [34]. A plot of initial kob, versus diethylpyrocarbonate concentration was linear (Fig. 3 B) and a In/ln plot of the same data also gave a straight line with a slope of 1.08 (Fig. 3C), suggesting that the inactivation was due to the interaction of diethylpyrocarbonate with one essential histidyl residue. The second-order rate constant was k , = 13.4 M-' min-'.

'

pM

pmol. min- I . ng-

356.7 f 16.1 185.7 f 14.2 154.3 18.3 193.0 -t- 23

0.346 f 0.09 0.396 f 0.058 0 218 & 0.08 0.132 f 0.030

The pH dependence of inactivation was examined using 10 mM diethylpyrocarbonate in 0.1 M phosphate between pH 5.9 and 8.5 at 25°C. Initial rates of inactivation increased with increasing pH up to pH 7.2. The pK, of the reactive group was determined by plotting the log of the second-order rate constant for inactivation, k l , against pH. The plot showed a typical titration curve with a pK, of 6.4 (Fig. 4). Since diethylpyrocarbonate reacts only with the unprotonated form of imidazole in proteins [35] and the pK, value of 6.4 was within the range expected for a histidine residue, the results are consistent with the proposal that the inactivation results from the modification of one essential histidine. Although diethylpyrocarbonate can also modify lysyl, sulfhydryl and tyrosyl residues, the reagent is reasonably specific for reaction with histidine in the pH range 5.5-7.5 [36]. In addition, diethylpyrocarbonate inactivation was totally reversed by a 1-h incubation with 0.2 M hydroxylamine at 25°C at pH 7.2, a molecule which removes the N-carbethoxyl groups from modified histidines and tyrosines but not from the other amino acids [37], thus eliminating lysyl and sulfhydryl modification as a basis for the loss of activity. Treatment of aminopeptidase N with 10 mM diethylpyrocarbonate for 1 h resulted in an increase of absorbance between 230 - 250 nm, which is characteristic of histidine modification [36], but no significant change in the absorbance was detected at 278 nm (Fig. S), indicating that the tyrosines were not affected by the reagent under the conditions used [38]. The increase in absorbance at 240 nm indicated the carbethoxylation of one histidyl residue, based on the value

389

-

20

2.0

2z 1.6

15-

._ t E

a

5

W

Y

2

cr >fr

1.2 5-

0.8

2tR

10-

r

0 5

7

8

9

PH

0.4

3 0

1 6

I

10

2b

$0

I

40

I

50

TIME min

Fig. 4. Elfect o f p H on inactivation by diethylpyrocarbonate. The enzyme (1 pg) was incubated with 10 mM diethylpyrocarbonate in 0.1 M potassium phosphate at 25°C at pH values indicated. The second-order rate constants ( k , ) were calculated by dividing the pseudo-first order constants kobs (determined as in Fig. 2A) by the concentration of diethylpyrocarbonate used (10 mM). The theoretical curve drawn assumes a pK, of 6.4

2

4 8

1.6 1.2 0.8 0.4 0

[DIETHYLPYROCARBONATE] mM Fig. 3. Inactivation of aminopeptidase N by diethylpyrocarbonate. (A) Plot of the log of residual activity against time at varying concentrations of diethylpyrocarbonate. The enzyme (1 pg) was incubated with 2 mM ( O ) , 5 mM (0),10 mM (B),13.8 mM ( A ) , 20 mM (A) and 25 mM (El) diethylpyrocarbonate in 0.1 M potassium phosphate pH 7.2 at 25°C. Enzyme activity was determined at regular intervals. The first-order rate constants kobs were calculated from the slopes of the straight lines. (B) Dependence of kobson [diethylpyrocarbonate]. Kobsfor inactivation was proportional to the concentration of diethylpyrocarbonate and the second-order rate constant kl was calculated from the slope of the line. (C) In kobsplotted against In [diethylpyrocarbonate]. The slope of the line gives the reaction order with respect to diethylpyrocarbonate

230 240 250 260 270 280 290 300 310

WAVELENGTH (nm) Fig. 5 . Ultraviolet absorption spectra of unmodfied and diethylpyrocarbonate-treated aminopeptidase. 2 mg aminopeptidase N in a 1.0-ml reaction mixture was incubated in the absence (1) or in the presence (2) of 10 mM diethylpyrocarbonate at 25°C in 0.1 M phosphate for I h and the reaction stopped by gel filtration before monitoring the spectra. The difference spectrum is shown in the inset

Effect of N-acetylimidazole treatment on aminopeptidase N activity

On reaction of aminopeptidase N with 10 mM Nacetylimidazole at 25 “C, pH 7.4, enzyme activity was only of E~~~ = 3200 M-’ cm-’ for N-carbethoxyhistidine. Thus, decreased to about 30% of the control in 1 h, although 5 mM the inactivation of aminopeptidase N could be attributed to N-acetylimidazole rapidly inactivates enzymes with essential tyrosine groups such as carboxypeptidase A 1391 and the modification of an essential histidine residue. The modification of the histidine by diethylpyrocarbonate angiotensin-converting enzyme [40]. No further decrease ocwas totally prevented by preincubation with 2 mM bestatin curred if the reaction was allowed to continue. Measuring and inhibitor 1,whilst inhibitor 2 had only a weak effect (14% enzyme activity in Hepes buffer instead of Tris buffer did protection, Table l), suggesting that the modified histidine is not change the rate of inhibition although Tris is known to probably located in the S1 subsite. Diethylpyrocarbonate at a deacetylate O-acetyltyrosine [41]. A large excess of Nconcentration of 10 mM increased the K , for [Leu5]- acetylimidazole (30 mM) totally abolished enzyme activity in 1 h. The kinetics of inhibition of aminopeptidase N are shown enkephalin threefold, after a l-h incubation, whilst the V,,, was unchanged (Table 2), indicating that the histidine might in Fig. 6. Semilog plots of residual activity versus time were linear, indicating that inactivation was due to a single chemical be important in substrate binding.

390

2 1.6

lj

1.2

20

0.8

i-'\J. -0015

0.4 0

230 240 2 5 0 260 2 7 0 280 290 300 3 1 0

TIME

min

WAVELENGTH (nm) Fig. 7. Ultraviolet absorption spectra of ttnmodified arid acetyluted uminopeptiduse N . 2 mg aminopeptidase N in a 1.O-ml reaction mixture was incubated in the absence (1) and in the presence of 30 mM (2) and 20 mM (spectrum not shown) N-acetylimidazole in 50 mM Hepes pH 7.5 for 1 h; reactions were stopped by gel filtration. The modified enzyme retained 30% and 0% of its original activity for 20 mM and 30 mM N-acetylimidazole, respectively. The difference spectra for 20 m M (V)and 30 mM ( A )N-acetylimidazole are shown in the inset

[ N ACETYL IMIDAZOLE] rnM Fig. 6. Inactivation of atninopeptidausc~N by N-acetyliiniduzole. (A) Effect of various concentrations of N-acetylimidazole on the rate of inactivation ofaniinopeptidase N . The enzyme (1 pg) in 50 mM Hepes p H 7.4 was treated with : 0 ( O ) ,5 mM (0),10 m M (W), 15 m M ( A ) , 20 m M (+), 25 mM (A)and 30 mM (0) N-acetylimidazole at 25°C. Aliquots were removed at regular intervals for measurement of the residual enzymc activity. Thc first-order rate constants kohswere determined from the slopes of the lines. (B) Dependence of kobson [Nacetylimidazole]. The plot shows the linear dependence of kobson Nacetylimidazole Concentration and the second-order rate constant k , was calculated from thc slope of the line. (C) In kohs plotted against In [N-acetylimidazole]. The slope of the line gives the reaction order with respect to N-acetylimidaxole

acetyltyrosine upon treatment with N-acetylimidazole is shown in Fig. 7. The modified enzyme showed a decrease in absorbance at 278 nm, a wavelength that corresponds to that of 0-acetyltyrosine = 1160 M - ' min-') and indicates the acetylation of 0.7 mol tyrosine/mol with 20 mM reagent and 1.05 mol/mol with 30 mM reagent. The partial inactivation of aminopeptidase N by N-acetylimidazole can therefore be attributed to the 0-acetylation of a tyrosine. The modification of this tyrosine was completely inhibited by the presence of 2 mM bestatin and inhibitor 2 while inhibitor 1 had only a partial protection effect (50% protection, Table l), suggesting the presence of the tyrosine in the S; subsite of the active site. N-Acetyliinidazole at a concentration of 20 mM had a mixed effect on the kinetic parameters after a 1-h incubation, doubling the K, and halving the VmaX (Table 2 ) , suggesting that the modified tyrosine could be involved in both substrate binding and the catalytic process. Inhibition of arninopeptidase N by carhoxyl group reagents

The activity of aminopeptidase N was rapidly but only partially diminished by the carboxyl group reagent EDC, at pH 6 for 30min (data not shown). This inhibition did not require addition of a nucleophile but was reversed at neutral event (Fig. 6A). The second-order rate constant for inacti- pH. This is in agreement with the formation of 0-acylisourea vation calculated from the slope of the replot of /cobsversus N- which is the result of a reaction of a carboxylic acid with the acetylimidazole concentration was 1.94 M min- (Fig. 6B). water-soluble carbodiimide [42]. The rapid reaction of EDC The ln/ln replot of the same data had a slope of 0.92 (Fig. 6C). with proteins is normally confined to carboxyl groups, at Thus, it could be concluded that the fixation of one molecule least when thiol groups are absent. Phenolic hydroxyl groups of N-acetylimidazole 'molecule enzyme leads to its partial inac- sometimes also react, but more slowly [43]. The rapid inactitivation. N-acetylimidazole can acetylate tyrosyl, thiol and vation here suggests a reaction with carboxyl residues. Activity lysyl groups [41]. However, the activity of aminopeptidase N could not be restored by the addition of 0.2 M hydroxylamine incubated with 20 mM N-acetylimidazole for 1 h was totally for 4 h, thus indicating that tyrosines were not involved in the restored by 0.2 M hydroxylamine in 50 min, indicating that reaction with EDC. Moreover, lysine. cysteine and threonine the loss of activity was not due to the modification of lysyl modifications are unlikely under the experimental conditions or cysteinyl residues Another proof of the formation of 0- of pH and temperature used [43].

391

0.003

-

0.002

'C .-

E

$

y

0.001

In order to confirm the role of a carboxyl group in the activity of the enzyme, another specific covalent reagent was used. Diazoacetamide has been shown to react almost exclusively with carboxylate groups to form glycolamide ester linkages [26]. Because of its high reactivity with almost all anions, diazoacetamide was used in bidistilled water (pH 6.1). 0.5 M diazoacetamide inhibited 60 - 70% of the enzymatic activity in 4 h. The reaction was very slow and the second-order rate constant kl given by the plot of the first-order rate constant kobsas a function of diazoacetamide concentration was 0.0024 M - l min-' (Fig. SA). The ln/lnplot ofkOb,versus diazoacetamide concentration gave a straight line with a slope of 0.99, indicating the modification of one carboxyl residue (Fig. 8 B). Bestatin and the other two inhibitors at a concentration of 2 mM were found to afford complete protection against inactivation by both diazoacetamide (Table 1) and EDC (data not shown). This suggests that the modified carboxyl group is localized near the zinc binding site, since the three compounds have a part in common which interacts with the metal ion (Fig. 1). When the ICs0 values of the inhibitors 1and 2 were measured for both diazoacetamide-treated and control enzyme, only the IC50of inhibitor 2 was found to be altered, by a factor 10, by the modification (not shown). These results are in agreement with the localization of the modified carboxyl group near both the zinc binding site and the S; subsite. Diazoacetamide treatment lead to a twofold increase in the K, of [Leu5]enkephalin with an insignificant decrease in V,,, (Table 2). This is consistent with a role of the carboxyl group in substrate binding. DISCUSSION Few studies have been devoted to the characterization of the active site of aminopeptidase N. Previous experiments have shown that diazonium[1HItetrazole modifies five histi-

dine residues and tetranitromethane five tyrosine residues, leading to loss of activity but no evidence was obtained for the presence of any of these residues at the active site [44,45]. The active site of an aminopeptidase of procaryotic origin (Aeromonas proteolytica) has been characterized using chemical reagents and the data have been interpreted by the involvement of one tyrosyl and one carboxylic acid residue in the enzymatic activity [30, 461. In order to understand the molecular organization of the active site of hog kidney aminopeptidase N, we have investigated the nature of the amino acids involved in substrate binding or in the catalytic process by chemical modification experiments. The results of this study show that a single arginine is essential for enzymatic activity, and more precisely for substrate binding in aminopeptidase N. The presence of arginine residues in the active site of various zinc metallopeptidases and their role in substrate or inhibitor binding have been previously demonstrated. Butanedione was shown to interact with arginine residues involved in substrate binding and located in the active site of neutral endopeptidase 24.1 1 [47,48]. In carboxypeptidase A, the position of the guanidinium group of Arg145 is such that it interacts with the carboxy-terminal group of the substrates [49]. In contrast, in thermolysin, Arg203 has been shown to form a hydrogen bond with the carbonyl of the Pi-Pl, amide bond of the substrate, allowing the binding of an extended peptide in agreement with the endopeptidase nature of this peptidase [24]. In aminopeptidase N, the low (40-50%) protection provided by bestatin and inhibitor 2 against butanedione modification suggests that the functional arginyl residue may be located in the Sl, subsite, probably interacting with the Pl-P2 or the P2-Pj amide bond of substrates. This is consistent with the fact that aminopeptidase N is more sensitive to inhibition by tripeptides than dipeptides and that this enzyme hydrolyzes tripeptides or larger peptides more efficiently than smaller peptides (review in [17]). This arginine could well correspond to Arg441 of rat kidney aminopeptidase N which has been suggested to align with Arg203 of thermolysin [21]. The presence of an essential histidyl residue in aminopeptidase N is perhaps unexpected, since no functional histidine was found at the active site of Aeromonas aminopeptidase [46]. However, histidines have been found to participate in catalysis of neutral endopeptidase [50, 511, thermolysin [52] and other neutral Lacterial proteases [53]. In exopeptidases such as carboxypeptidase A, the corresponding residue was shown to be a tyrosine [39]. Recent calculations, based on crystallographic studies, have suggested that the NH of the essential histidine in thermolysin (His231) is important in substrate binding [24]. The steady-state kinetics here indicate that the essential histidyl residue in aminopeptidase N is also mainly involved in substrate binding. In addition, its likely location in the S1 subsite could be favorable for this histidine to play a similar role to His231 of thermolysin. Watt and Yip [21] have, however, suggested that a tyrosyl residue in aminopeptidase N might also fulfill this role (Tyr476). The results also show the presence of a tyrosine in the active site of aminopeptidase N. The slight effect of Nacetylimidazole on the enzymatic activity is similar to that observed with thermolysin [54] and suggests that either this tyrosine is poorly accessible or weakly reactive because of its micro-environment, or that this residue is not essential in catalysis. Crystallographic studies of thermolysin in the presence of a phosphoramidate inhibitor have shown that, in addition to His231, a tyrosine (Tyr157) participates in the stabilization of the tetrahedral transition state through hydro-

392 gen bond formation [24]. It seems reasonable to expect that the modified tyrosine found in the S; subsite of aminopeptidase N plays such a role, since its acetylation resulted in a slight decrease in V,,, and a slight increase in K, for [Leujlenkephalin. Similar results have been obtained for Aeromonas aminopeptidase [46]. A single functional acidic residue was also found to be involved in substrate binding. Two essential carboxyl groups could be assumed to be present in the active site of an aminopeptidase. The first, located in the catalytic site, would correspond to the carboxyl group of the glutamate found in the consensus sequence, Val-Xaa-Xaa-His-Glu-Xaa-Xaa-His, whose counterparts in thermolysin (Glu143) and in neutral endopeptidase (Glu585) have been shown to be involved in the catalytic process [24, 551. The relatively small change in the enzymatic properties of aminopeptidase N following diazoacetamide treatment is therefore hardly reconcilable with a modification of the corresponding glutamate of this enzyme, especially as the K, and not the V,,, was altered. Nevertheless, a possible action of the glycolamide formed by modification of this glutamate, as a promotor of proton abstraction from HzO, cannot be completely ruled out. The second carboxyl group could be located at the end of the S1 subsite, accounting for the exopeptidase activity of aminopeptidase N, due to its interaction with the NH2 terminal of peptide substrates or inhibitors. This is supported by the findings that recognition of the S1 subsite of aminopeptidase N by substrates or inhibitors is critically dependent on the presence of a positively charged free amino group (171. It is unlikely that diazoacetamide reacted with this acceptor group, as the reagent increased only the ICjo of inhibitor 2 which interacts with the S; subsite of aminopeptidase N . These results, taken together with those of the protection experiments, are in agreement with the localization of the modified carboxyl group near both the zinc binding site and the S\ subsite. The proposed amino-group acceptor of the S1 subsite is probably located in a sterically hindered environment, so that EDC or diazoacetamide would not be able to react with it. Studies focused on the close correspondence between thermolysin and other zinc metallopeptidases in the location and function of mechanically important groups have shown a general conservation of active-site center geometries [24,49]. Our results show that the active site of aminopeptidase N shares common features with those of other zinc metallopeptidases, especially endopeptidases such as thermolysin. Although aminopeptidase N is an exopeptidase, the presence of an essential histidine in the S1 subsite together with an arginine in the S1-S; subsites involved in substrate binding and a tyrosine participating in the catalysis, suggests that its mechanism of action should be closely related to that of endopeptidases. These results should be taken into account for the design of either selective aminopeptidase blockers or mixed aminopeptidase N/neutral endopeptidase inhibitors (review in [16]). We are grateful to S. Turcaud for the synthesis of the specific inhibitors 1and 2 and P. Coric for the preparation of diazoacetamide. We thank C. Dupuis for typing the manuscript. This work is supported by the MinistPre de lu Recherche et de la Teclznologie (France).

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