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Analytical Biochemistry 292, 257–265 (2001)doi:10.1006/abio.2001.5083, available online at http://www.idealibrary.com on

Selective Neurotensin-Derived Internally QuenchedFluorogenic Substrates for Neurolysin (EC 3.4.24.16):Comparison with Thimet Oligopeptidase (EC 3.4.24.15)and Neprilysin (EC 3.4.24.11)

Vitor Oliveira,* Marcelo Campos,* Jefferson P. Hemerly,* Emer S. Ferro,†Antonio C. M. Camargo,‡ Maria A. Juliano,* and Luiz Juliano*,1

*Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Rua Tres de Maio,00, Sao Paulo, SP, 04044-020, Brazil; †Department of Histology, Institute of Biomedical Sciences,niversidade de Sao Paulo, Sao Paulo, 05508-900, SP, Brazil; and ‡Laboratory of Biochemistrynd Biophysics, Instituto Butantan, Sao Paulo, 05503-900, SP, Brazil

Received December 12, 2000; published online April 27, 2001

Internally quenched fluorescent peptides derivedfrom neurotensin (pELYENKPRRPYIL) sequencewere synthesized and assayed as substrates for neuro-lysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-di-nitrophenyl)ethylenediamine and Abz is ortho-amino-benzoic acid) was derived from neurotensin by theintroduction of Q-EDDnp at the C-terminal end of pep-tide and by the substitution of the pyroglutamic (pE)residue at N-terminus for Abz and a series of shorterpeptides was obtained by deletion of amino acids res-idues from C-terminal, N-terminal, or both sides. Neu-rolysin and TOP hydrolyzed the substrates at POY orYOI or ROR bonds depending on the sequence andsize of the peptides, while NEP cleaved P-Y or Y-Ibonds according to its S*1 specificity. One of these sub-trates, Abz-NKPRRPQ-EDDnp was a specific and sen-itive substrate for neurolysin (kcat 5 7.0 s21, Km 5 1.19

mM and kcat/Km 5 5882 mM21 z s21), while it was com-pletely resistant to NEP and poorly hydrolyzed byTOP and also by prolyl oligopeptidase (EC 3.4.21.26).Neurolysin concentrations as low as 1 pM were de-tected using this substrate under our conditions andits analogue Abz-NKPRAPQ-EDDnp was hydrolyzedby neurolysin with kcat 5 14.03 s21, Km 5 0.82 mM, andkcat/Km 5 17,110 mM21 z s21, being the best substrate sofar described for this peptidase. © 2001 Academic Press

1

To whom correspondence should be addressed. Fax: 55-11-5575-9040. E-mail: juliano.biof@epm.br.

0003-2697/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

Key Words: fluorogenic substrates; neurotensin; neu-rolysin; thimet oligopeptidase; neprilysin; prolyloligopeptidase.

Neurolysin (EC 3.4.24.16) is a zinc-dependent pepti-dase member of the metallopeptidase M3 family thathas in its primary sequence the HEXXH motif (1, 2),and was reported to present selectivity to oligopeptides(2, 3). Strict selectivity to oligopeptides has also beendescribed for thimet oligopeptidase (EC 3.4.24.15 orTOP)2 (4–6) a neurolysin-related peptidase that be-longs to the same family with 65% of homology in theamino acid sequence (1, 7). The designation oligopep-tidase has been used since 1979 (4) and, recently, mo-lecular mechanisms for oligopeptidase selectivity weredescribed for neprilysin (EC 3.4.24.11 or NEP) (8) andfor prolyl oligopeptidase (9–11), the 3D structures ofwhich were reported, showing how proteins are ex-cluded from the active sites. However, the mecha-nism(s) that permits to neurolysin and TOP to act onlyon oligopeptides remains unknown.

2 Abbreviations used: TOP, thimet oligopeptidase; NEP, neprily-sin; NT, neurotensin; MCC, 3-carboxy-7-methoxycoumarin; Dnp, 2,4-dinitrophenyl; Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitro-phenyl)ethylenediamine; Tris, Tris(hydroxymethyl)aminomethane;MALDI-TOF, matrix-assisted laser desorption ionization time offlight; HPLC, high-performance liquid chromatography; DTT, DL-

dithiotreitol; Suc, N-succinyl; MCA, 7-methoxycoumarin-4-acetyl;AUF, arbitrary units of fluorescence; TFA, trifluoroacetic acid.

257

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1

7

adw

pe

P

pm(w

258 OLIVEIRA ET AL.

Neurolysin is widely distributed in mammalian tis-sues and is thought to be involved in the inactivationand/or generation of a great number of biologicallyactive peptides (12). Prolyl oligopeptidase, NEP, TOP,and neurolysin are able to hydrolyze the biologicallyactive peptide neurotensin (NT) in vitro and they couldbe participating in the catabolism of this biologicallyactive peptide (13–16). Furthermore, in vivo experi-ments have shown that NT degradation can be blockedby TOP and neurolysin inhibitors (17) and a highlyspecific neurolysin inhibitor potentiated the NT-in-duced antinociception of mice in the hot plate testwhen administrated intracerebroventricularly (18).

Neurolysin and TOP hydrolyze many biologically ac-tive oligopeptides at the same peptide bond, suggestingthat they have similar active centers (19–24). On theother hand, NT is hydrolyzed at different bonds bythese peptidases: TOP hydrolyzes the ROR bond, pro-

ucing NT1–8 and NT9–13, and neurolysin cleaves thePOY bond, producing NT1–10 and NT11–13 (25). Thisfeature has been used to distinguish the neurolysinfrom TOP activity using NT as substrate; however, itrequires discontinuous assays followed by HPLC anal-ysis and it is not capable of distinguishing the NEPactivity that also produces NT1–10 and NT11–13 whichcauses the necessity of complementary addition of spe-cific inhibitors (12, 26). A continuous assay for neuro-lysin could be performed using the internally quenchedfluorogenic substrate QFS (MCC-Pro-Leu-Gly-DLys-Dnp) (12, 24) with a kcat/Km of 440 mM21 z s21 for apurified neurolysin from rat liver. However, this sub-strate was initially developed for TOP having kcat/Km of010 mM21 z s21 for its recombinant form (27), therefore

a specific inhibitor is required to distinguish neuroly-sin from TOP (12, 26).

In the present work, using the human NT sequenceas model, we developed selective fluorogenic substratesfor neurolysin that distinguished it from TOP andNEP. The obtained internally quenched fluorescentpeptides are the most sensitive and selective sub-strates for neurolysin so far described.

MATERIALS AND METHODS

Neurolysin

The recombinant cDNA of porcine kidney neurolysin(cytosolic form) was a kind gift from Drs. S. Hirose andA. Kato (Department of Biological Sciences, Tokyo In-stitute of Technology, Yokohama, Japan). The proce-dures for expression and purification of recombinantporcine kidney neurolysin were performed as previ-ously described (25).

Thimet Oligopeptidase

The purified recombinant rat testes TOP (rTOP) wasobtained as previously described (28). The amino acid

Pi

composition of the purified enzyme [Asp, 57; Glu, 97;Ser, 32; Gly, 46; His, 16; Arg, 38; Thr, 35; Ala, 58; Pro,34; Tyr, 18, Val, 43; Met, 18; Ile, 18; Leu, 71; Phe, 27;Trp (not determined); Lys, 41] reproduced the theoret-ical composition values of the rat testes TOP (29). Thehomogeneity of the enzyme preparation was confirmedby MALDI-TOF mass spectrometry (TofSpec-E, Micro-mass, Manchester, UK). Calculated MW, MH1 58,315; obtained MW, MH1 5 79,748. A difference of

approximately 2% is a reasonable error for the lowaccuracy condition for the external calibration em-ployed. The purified enzyme was aliquoted in vialscontaining about 1 mg of enzyme in 2% serum albuminand 30% glycerol. After an initial reduction in 15% theenzyme activity remained constant for at least 2months at 270°C.

The enzyme concentration was determined by aminocid analysis corrected for the 15% enzyme activityecrease. We also performed an active site titration,ith a tightly biding inhibitor (Z-PheC(PO2CH2)-Ala-

Lys-Ile), with a Ki ; 2 nM (30) that was a kind gift fromDr. V. Dive (Department d’Ingenierie et d’Etudes desproteins, DSV, Gif Yvette Cedex, France). For thistitration, aliquots of rTOP were preincubated at differ-ent inhibitor concentrations, for 24 h at 4°C, in a finalvolume of 100 mL in the TBS buffer, containing 30%glycerol and 1 mg/mL albumin, after which the remi-niscent activity was measured with 9 mM of the sub-strate Abz-GFSPFRQ-EDDnp, at a final volume of 1mL (dilution of 10 times) in TBS buffer at 37°C. DTTwas added 5 min before the preincubation and 5 minbefore the reminiscent activity determination; how-ever, DTT concentration was kept constant (0.5 mM)regardless of the dilution employed. The enzyme con-centration and the Ki was calculated according to themethod described by Knight (31). All the obtained datawere fitted to nonlinear least-square equations usingGrafit v 3.0 from Erithacus Software (32).

Neprilysin

The pure recombinant NEP was a kind gift fromProf. Dr. G. Boileau (Department of Biochemistry, Uni-versite de Montreal, Montreal, Quebec, Canada. The

rocedures for expression and purification are providedlsewhere (33, 34).

rolyl Oligopeptidase

Prolyl oligopeptidase from pig muscle was purified asreviously described (35). The active site titration wasade using the irreversible inhibitor Z-Pro-Prolinal

Sigma, St. Louis, MO) and the reminiscent activityas measured with the fluorogenic substrate Suc-Gly-

ro-MCA (Sigma) in 20 mM Tris–HCl, pH 7.5, contain-

ng 1 mM EDTA. The concentration of DTT was kept

259SELECTIVE SUBSTRATES FOR NEUROLYSIN

constant (10 mM) in all steps regardless the dilutions.The enzyme concentration was calculated plotting thereminiscent activity against the inhibitor concentra-tion. The intercept at the X-axis obtained by linearregression analysis gives the enzyme concentration(31).

Peptide Synthesis

The intramolecularly quenched fluorogenic peptidescontaining N-[2,4-dinitrophenyl]ethylenediamine (EDDnp)attached to glutamine were synthesized by solid-phasestrategy and the details of which are provided else-where (36). An automated bench-top simultaneousmultiple solid-phase peptide synthesizer (PSSM 8 sys-tem from Shimadzu) was used for the solid-phasesynthesis of all the peptides by the Fmoc procedure.The final deprotected peptides were purified by semi-preparative HPLC using an Econosil C-18 column (10mm, 22.5 3 250 mm) and a two-solvent system: (A) tri-fluoroacetic acid (TFA)/H2O (1:1000) and (B) TFA/acetonitrile (ACN)/H2O (1:900:100). The column waseluted at a flow rate of 5 ml/min with a 10% (or 30%)–50% (or 60%) gradient of solvent B over 30 or 45 min.Analytical HPLC was performed using a binary HPLCsystem from Shimadzu with a SPD-10AV ShimadzuUV–vis detector and a Shimadzu RF-535 fluorescencedetector, coupled to an Ultrasphere C-18 column (5mm, 4.6 3 150 mm) which was eluted with solventsystems A and B at a flow rate of 1 ml/min and a10–80% gradient of B over 20 min. The HPLC columneluates were monitored by their absorbance at 220 nmand by fluorescence emission at 420 nm following ex-citation at 320 nm. The molecular weight and purity ofsynthesized peptides were checked by MALDI-TOFmass spectrometry (TofSpec-E, Micromass) and/or pep-tide sequencing using a protein sequencer PPSQ-23(Shimadzu Tokyo, Japan). The purity of the synthe-sized peptides were, at least, 95%.

Hydrolysis of NT

NT was purchased from Sigma. The reactions of NTwith neurolysin and TOP were carried out at 37°C in50 mM Tris–HCl buffer, pH 7.4, containing 100 mMNaCl or at 37°C, 50 mM Tris–HCl buffer, pH 8.0, forNEP. The 30 mM NT was incubated under the condi-tions described above with 0.128 nM neurolysin; 0.488nM TOP, or 0.286 nM NEP. TOP assays require pre-incubation of the enzyme with 0.5 mM DTT for enzy-matic activation, which is added to the cuvette contain-ing the appropriate buffer. For initial velocitiesmeasurements, aliquots were taken from the reactionmixture every 5 min until 30 min; the reactions were

stopped by adding an acidic solution (HCl 10%) andfreezing. Standard curves of NT based on the area of

the peak in the UV–visible detector of the HPLC at 280nm were generated and used to quantify the hydrolysisproducts of the assays. Each aliquot were then ana-lyzed by HPLC, monitoring the absorbance at 280 nmand the area of the product plotted against the time ofincubation. The velocity was calculated determiningthe slope by linear regression analysis.

Kinetic Assays

Hydrolysis of the fluorogenic peptidyl substrates at37°C in 50 mM Tris–HCl buffer, pH 7.4, containing 100mM NaCl (neurolysin and TOP) or 50 mM Tris–HCl,pH 8.0 (NEP), was followed by measuring the fluores-cence at lem 5 420 nm and lex 5 320 nm in a HitachiF-2000 spectrofluorometer. The 1-cm-path-length cu-vette containing 2 ml of the substrate solution wasplaced in a thermostatically controlled cell compart-ment for 5 min before the enzyme solution was addedand the increase in fluorescence with time was contin-uously recorded for 5–10 min. Preceding this step forthe TOP assays a preincubation of the enzyme with 0.5mM DTT for activation was applied in the cuvettecontaining 2 mL of the appropriate buffer. The slopewas converted into moles of hydrolyzed substrate perminute based on the fluorescence curves of standardpeptide solutions before and after total enzymatic hy-drolysis. The concentration of the peptide solutionswas obtained by colorimetric determination of the 2,4-dinitrophenyl group (17,300 M21 z cm21 extinction co-efficient at 365 nm). The enzyme concentration forinitial rate determination was chosen at a level in-tended to hydrolyze less than 5% of the substratepresent. The inner-filter effect was corrected using anempirical equation as previously described (37). Thekinetic parameters were calculated according Wilkin-son (38) as well as by using Eadie–Hofstee plots. Allthe obtained data were fitted to nonlinear least-squareequations, using Grafit v 3.0 from Erithacus Software(32).

Determination of Cleaved Bonds

The cleaved bonds were identified by isolation of thefragments by HPLC and the retention times of theproducts fragments were compared with authentic syn-thetic sequences and/or by molecular weight, whichwas determined by MALDI-TOF mass spectrometryand/or by peptide sequencing, using a protein se-quencer PPSQ-23 (Shimadzu Tokyo, Japan).

Amino Acid Analysis

The amino acid compositions, the concentration ofthe peptides, and the purified rTOP were determined

as follows: the samples were digested for 22 h at 110°Cin 6 N HCl containing 1% phenol in vacuum-sealed

R

H

fhcKLttAIn

p(

N

scnbobtah(bptt

Rsfcs

V

V

260 OLIVEIRA ET AL.

tubes and then subjected to amino acid analysis usinga pico-Tag station (39).

RESULTS

Hydrolysis of NT by Neurolysin, TOP, and NEP

The relative rates of hydrolysis of human NT (pELY-ENKPRRPYIL) by neurolysin, TOP and NEP are re-spectively 100, 27, and 84% under our conditions (ini-tial velocities measurements and saturating substrateconcentration, 30 mM of NT). The obtained cleavagesites are in accordance as previously described results(20, 25), namely, neurolysin at POY bond; TOP atOR bond and for NEP at POY and YOI bonds.

ydrolysis of Synthetic Fluorogenic QuenchedPeptides Based on NT

Table 1 shows the fluorogenic substrates derivedrom NT sequence and the velocities obtained for theirydrolysis by neurolysin, TOP, and NEP, and also theleavage points determined. The substrate Abz-LYEN-PRRPYILQ-EDDnp (peptide I) differs from NT (pE-YENKPRRPYIL) by the introduction of Q-EDDnp athe C-terminal end of peptide and the substitution ofhe pyroglutamic (pE) residue at N-terminus by Abz.ll other peptides derived from Abz-LYENKPRRPY-

TAB

Maximum Velocities for Hydrolysis by Neurolysin, TO(pELYENKPRRPYIL) and Det

No. Abz-peptidyl-Q-EDDnp

Neurolysin (24.16)

Vmax/[E] (s21)a Cleavage site

I LYENKPRRPYIL 1.5 P 2 Y 65%Y 2 I 35%

II KPRRPYIL 0.4 P 2 Y 37%Y 2 I 63%

III ENKPRRPYIL 1.6 P 2 Y 36%Y 2 I 64%

IV ENKPRRPYI 2.3 P 2 Y 79%Y 2 I 21%

ENKPRRPY 0.8P 2 Y 16%Y 2 Q 40%R 2 R 44%

I LYENKPRRP 3.8 R 2 RVII ENKPRRP 1.4 R 2 RVIII NKPRRP 7.0 R 2 RIX KPRRP 2.6 R 2 RX PRRP Resistantb —XI GGFLPRRP 1.8 R 2 R

a Overall velocity estimated from the hyperbolic plot of V versus [is the substrate concentration.

b Peptides were resistant to hydrolysis until 8 nM of enzymes.

LQ-EDDnp by deletion of amino acids from C-termi-al, N-terminal, or both sides. The only exception is

eptide XI that was designed as a hybrid of enkephalinGGFL) and NT (PRRP) segments.

eurolysin

Neurolysin was able to hydrolyze all the obtainedynthetic internally quenched fluorescent peptides ex-ept the shortest of the series (peptide X). In contrast toatural neurolysin that was cleaved only at the POYond, the peptides I to IV were hydrolyzed at the POYr YOI bonds. The preferential hydrolysis at the POYond in peptide I changed to YOI removing two orhree amino acids from the N-terminus (peptides IInd III) and the preference returned to POY bondydrolysis by additional removing Leu at C-terminuspeptide IV). An unusual result was the three cleavedonds at POY, YOQ, and ROR that were observed ineptide V, which in comparison to largest peptide inhe series has two amino acids deleted from each ex-remity.

The peptides VI to IX were hydrolyzed only at the2R bond and they have in common the C-terminalequence KPRRPQ-EDDnp. The kinetic parametersor the hydrolysis by neurolysin of these peptides in-luding the hybrid peptide XI were determined and arehown in Table 2. The higher kcat/Km value was ob-

served with the hydrolysis of Abz-LYENKPRRPQ-

1

and NEP of the Peptides Derived from Neurotensinination of the Cleavage Sites

TOP (24.15) NEP (24.11)

Vmax/[E] (s21)a Cleavage site Vmax/[E] (s21)a Cleavage site

0.3 P 2 Y 68% 0.8 P 2 Y 50%Y 2 I 32% Y 2 I 50%

Resistantb — 0.3 P 2 Y 60%Y 2 I 40%

Resistantb — 12 P 2 Y 53%Y 2 I 47%

0.9 R 2 R 84% 1.4 P 2 Y 25%P 2 Y 16% Y 2 I 75%

1.1 R 2 R 3.0 P 2 Y

0.3 R 2 R Resistantb —3.2 R 2 R Resistantb —0.7 R 2 R Resistantb —3.7 R 2 R Resistantb —

Resistantb — Resistantb —3.7 R 2 R Resistantb —

where V is the velocity measured in the spectrofluorometer and [S]

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P,erm

S],

EDDnp (peptide VI). The deletion of N-terminal dipep-tide LY resulted in peptide Abz-ENKPRRPQ-EDDnp

frLtoTwlTTRptototAEs

tttppp

P

ahtqn

(

H

tbl(

261SELECTIVE SUBSTRATES FOR NEUROLYSIN

(VII) that was the poorest substrate in this series. Thedeletion of Glu, resulting in Abz-NKPRRPQ-EDDnp(peptide VIII), restored the susceptibility to neurolysin,indicating that Glu residue has an unfavorable effectthat seems to be compensated by Leu and Tyr in pep-tide VI. A further shortening by deletion of Asn re-sulted in Abz-KPRRPQ-EDDnp (peptide IX) that wasmore resistant to neurolysin, but still better hydro-lyzed than peptide VII. The hybrid peptide (IX) waswell hydrolyzed, confirming the influence of the aminoacids in the nonprime site of the substrates on itssusceptibility to neurolysin.

TOP

In contrast to hydrolysis of natural NT by TOP inwhich the ROR bond is hydrolyzed, the peptide Abz-LYENKPRRPYILQ-EDDnp (peptide I, Table 1) washydrolyzed at the POY or YOI bonds with preferenceor the latter bond, similar to that observed with neu-olysin. The deletion of N-terminal sequence LYE orY turns the corresponding peptides (II and III) resis-ant to TOP. However, the deletion of C-terminal Leur sequence YIL resulted in substrates susceptible toOP (peptides IV to IX); some of them were hydrolyzedith a velocity higher than that observed with neuro-

ysin, but the cleaved bond moved to the ROR bond.able 2 shows the kinetic parameters for hydrolysis byOP of the peptides that were hydrolyzed only at theOR bond by neurolysin as well as by TOP whichermits a more accurate comparison between the ac-ivities of the two peptidases. All the kcat/Km valuesbtained with TOP were one or two orders of magni-ude lower than those obtained with neurolysin. Thenly exception was the hybrid peptide (XI) which washe best substrate for TOP in the series. The substratesbz-LYENKPRRPQ-EDDnp and Abz-NKPRRPQ-

TAB

Kinetics Parameters for Hydrolysis by Neurolysin anfrom NT That Were Hyd

No. Abz-peptidyl-Q-EDDnp

Neurolysin (24.16)

k cat

(s21)Km

(mM)k

(mM

VI LYENKPR 2 RP 3.8 6 0.1 0.44 6 0.03VII ENKPR 2 RP 1.35 6 0.07 5.8 6 0.6VIII NKPR 2 RP 7.0 6 0.2 1.19 6 0.09IX KPR 2 RP 2.63 6 0.07 3.3 6 0.2XI GGFLPR 2 RP 1.77 6 0.04 0.47 6 0.02

Note. 2 indicates the cleavage site.

DDnp (peptides VI and VIII, Table 2) have the higherelectivity to neurolysin relative to TOP.

pT

NEP

NEP cleaved only Abz-LYENKPRRPYILQ-EDDnp(peptide I, Table 1) and its lower homologues, in whichthe hydrophobic C-terminal amino acid Y or segmentsYIL or YI were maintained (peptides II to IV). Thecleavage sites of these peptides were at the POY orYOI bonds, which is in accordance to the well-knownspecificity of NEP at P91 for aromatic and hydrophobicresidues (40, 41). There was no clear preference forhydrolysis of POY or YOI bonds except in peptide IVhat the YOI bond is preferred for hydrolysis and pep-ide V that was cleaved only at the POY bond and washe most susceptible to NEP in the series. All othereptides were resistant to NEP, including the hybrideptide (XI), although peptides VI and XI have hydro-hobic amino acids in their sequences.

rolyl Oligopeptidase

Peptide VIII was assayed with prolyl oligopeptidases described in the kinetic assays section for TOP;owever, the buffer was 20 mM Tris–HCl, pH 7.5, andhe concentration of DTT used for activation (also re-uired for prolyl oligopeptidase) was 10 mM. The ki-etic parameters obtained were k cat 5 0.03 s21, Km 5

0.44 mM, and k cat/Km 5 68 mM21 z s21 and although twoproline residues were present in the peptide VIII se-quence, the unique cleavage site detected was at POQAbz-NKPRRPQ-EDDnp).

ydrolysis of the Peptides Derived from Abz-NKPRRPQ-EDDnp Scanned by Ala

To obtain more information on the determinants ofhe enzyme–substrate interactions that are responsi-le for the differential recognition by TOP and neuro-ysin, we took the substrate Abz-NKPRRPQ-EDDnppeptide VIII) as reference and synthesized a series of

2

TOP of the Fluorogenic Quenched Peptides Derivedyzed Only at ROR Bond

TOP (24.15)Selectivity:

k cat/Km 16

k cat/Km 15

Km

z s21)k cat

(s21)Km

(mM)k cat/Km

(mM21 z s21)

6 0.30 6 0.03 7 6 1 43 2013 3.2 6 0.1 21 6 1 152 1.52 0.70 6 0.02 12.1 6 0.7 58 1017 3.7 6 0.7 114 6 25 32 256 3.7 6 0.1 2.8 6 0.3 1321 2.9

LE

drol

cat/21

86323

58879

376

eptides changing each residue of the sequence by Ala.he peptide VIII was chosen due to its high selectivity

ddP

cw

t

VXXXXXX

c

262 OLIVEIRA ET AL.

to neurolysin in terms of kcat/Km ratio relative to TOPand was hydrolyzed by neurolysin with the highestcatalytic constant (k cat 5 7.0 s21, Table 2). The kineticparameters for the hydrolysis by TOP and neurolysinof these peptides are presented in the Table 3. All theresultant substrates containing Ala were less selectivefor neurolysin relative to TOP in comparison with pep-tide VIII (Abz-NKPRRPQ-EDDnp) and all of themwere resistant to NEP. The kcat/Km values for the hy-

rolysis by neurolysin of the peptides containing Alaid not differ significantly from those of Abz-NK-RRPQ-EDDnp, except when Ala was located at P1 or

P91 position. Abz-NKPARPQ-EDDnp (peptide XV) washydrolyzed by neurolysin with kcat/Km value signifi-antly lower and Abz-NKPRAPQ-EDDnp (peptide XVI)as hydrolyzed with kcat/Km value significantly higher

in comparison with Abz-NKPRRPQ-EDDnp (peptideVIII). These differences are essentially due to the kcat

values, whereas the substrate with Ala at P91 position(peptide XVI, Abz-NKPRAPQ-EDDnp) was hydrolyzedwith a k cat 5 14 s21, being the best substrate so fardescribed for neurolysin.

TAB

Kinetics Parameters for Hydrolysis by Neurolysin anfrom Peptide V

No. Abz-peptidyl-Q-EDDnp

Neurolysin (24.16)

k cat

(s21)Km

(mM) (

III NKPR 2 RP 7.0 6 0.2 1.19 6 0.09II AKPR 2 RP 4.97 6 0.02 1.15 6 0.08III NAPR 2 RP 6.97 6 0.03 1.03 6 0.08IV NKAR 2 RP 5.76 6 0.01 1.38 6 0.08V NKPA 2 RP 1.23 6 0.002 0.56 6 0.03VI NKPR 2 AP 14.03 6 0.06 0.82 6 0.08VII NKPR 2 RA 1.24 6 0.003 0.25 6 0.01

Note. 2 indicates the cleavage site.

FIG. 1. Initial velocities of hydrolysis of 12 mM Abz-NKPRRPQ-EDDoncentrations. The linear fits with statistical weighting gave the slopes 49

All the peptides from this series were hydrolyzed byTOP with higher kcat/Km values than the reference pep-ide (VIII) and this occurs due to significant higher kcat

values (Table 3). As a consequence the selectivity of allthese peptides for neurolysin was reduced in compari-son with TOP.

Sensitivity of Abz-NKPRRPQ-EDDnp for NeurolysinActivity Detection

Figure 1 shows the initial velocities of the hydrolysisreactions of neurolysin (A) and TOP (B) at differentconcentrations with peptide VIII at a fixed concentra-tion (12 mM). The selectivity under this condition givenby the ratio between the slopes taken from the linearfits is 236 (neurolysin/TOP). Velocities lower than 5AUF/min fall into the noise level of the equipment(spectrofluorometer HITACHI F-2000 with the slope ofthe standard curve being 7500 AUF/mM for Abz-NKPR-OH) as can be observed in the lower velocitiesfor TOP (Fig. 1B). Thus, reliable activities were de-tected as low as 1 pM of neurolysin under our condi-

3

TOP of the Fluorogenic Quenched Peptides DerivedI by Ala Scan

TOP (24.15)Selectivity:

k cat/Km 16

k cat/Km 15

cat/Km21 z s21)

k cat

(s21)Km

(mM)k cat/Km

(mM21 z s21)

5,882 0.70 6 0.02 12.1 6 0.7 58 1014,322 0.75 6 0.01 10.2 6 0.3 74 586,767 3.6 6 0.2 7.1 6 0.8 507 134,174 8 6 1 64 6 7 125 332,196 4.2 6 0.2 36 6 3 117 197,110 8.7 6 0.5 15 6 1.5 580 304,960 5.7 6 0.2 12.4 6 0.5 460 11

LE

dII

kmM

1

np (peptide VIII) by neurolysin (A) and TOP (B) under different58 6 95 AUF/nM for neurolysin (A) and 21 6 1 AUF/nM for TOP (B).

rtttwtdtb

b

ecYotc

p

uc2ett

dmeWGr

263SELECTIVE SUBSTRATES FOR NEUROLYSIN

tions ([S] 5 10 3 Km). On the other hand, as indicatedby the ratio between the slopes, 240 times more TOP(0.24 nM) was the minimum enzyme concentrationrequired for the activity detection in the same sub-strate concentration ([S] ; Km). The pH-curve profile(not shown) demonstrated that the best pH for neuro-lysin hydrolytic activity on Abz-NKPRRPQ-EDDnpwas in the range between 7.5 and 8.0.

DISCUSSION

The susceptibility of the peptides to neurolysin, TOP,and NEP presented in Table 1 shows unexpected re-sults concerning the cleavage sites of the substrates.TOP cleavage site changed from ROR in the naturalNT to POY and YOI in the peptide I (Table 1). Neu-olysin cleavage site changed from POY in natural NTo ROR in the C-terminal shorted peptides VI–IX. Thewo most selective substrates for neurolysin were pep-ides VI and VIII, whose cleavage bond was RORhich is the TOP cleavage site at natural NT. Con-

rasting with this behavior, NEP representing a well-efined thermolysin-like primary specificity cleavedhe substrates (peptides I–V) at the same peptideonds as in NT, according to its described S91 specificity

(40, 41) as well as its preferential exopeptidase ratherthan endopeptidase activity (42–44) since peptidesI and VI were not hydrolyzed by NEP at the LOYond.Peptide I was susceptible to hydrolysis by TOP; how-

ver, a displacement of the cleavage point occurred inomparison to NT hydrolysis from ROR to POY orOI. On the other hand, the peptides II and III with-ut N-terminal extensions residues in relation to pep-ide I were resistant to hydrolysis by TOP. Taking theleavage at POY as P1OP91, the difference from pep-

tide I to peptide III is the absence of the residues atP8 and P9 positions. As previously described (45)TOP preferentially hydrolyzes its substrates near theC-terminus which is in accordance with the cleavagesite displacement from ROR in NT to POY and YOI in

eptide I; however, the requirement for residues at P8

and P9 for a detectable hydrolysis as observed withpeptide III was an unexpected observation.

The two most selective substrates for neurolysinwere peptides VI and VIII, since they were resistant toNEP and were very poor substrates for TOP as shownin Tables 1 and 2. Because the selectivity in terms ofkcat/Km ratio is of two orders of magnitude (Table 2), we

sed the peptide VIII sequence as reference because itombined selectivity and the highest sensitivity (Tableand Fig. 1) to synthesize a series of peptides changing

ach residue by Ala (Table 3). Each position was foundo be important for the selectivity of neurolysin relative

o TOP, particularly the positions P3 and P92, in which

Ala reduced the selectivity obtained with the reference 1

substrate (peptide VIII) almost 10 times, from 101 to13 and 11, respectively. It is noteworthy that the highselectivity of peptide VIII containing Pro at P2 and P92match exactly with the most selective inhibitors forneurolysin obtained by Jiracek and colleagues (46).

Peptide VIII was assayed with a purified prolyl oli-gopeptidase from pig muscle and the kinetic parame-ters obtained, especially the very low catalytic constantobtained (k cat 5 0.03 s21), showed that this peptide is avery poor substrate also for this enzyme. In conclusionwe obtained a very specific and sensitive substrate forneurolysin (peptide VIII, Abz-NKPRRPQ-EDDnp) aswell as the most susceptible substrate for neurolysinthus far described, particularly due to a high kcat value(peptide XVI, Abz-NKPRAPQ-EDDnp).

ACKNOWLEDGMENTS

This work was supported by the Fundacao de Amparo a Pesquisao Estado de Sao Paulo (FAPESP), Conselho Nacional de Desenvolvi-ento Cientıfico e Tecnologico (CNPq), and Financiadora de EstudosProjetos (FINEP) through the PADCT-Biotecnologia III program.e acknowledge the excellent technical assistance of Mrs. Eglelisa. Andrade and also Dr. Fernanda V. Portaro for providing pure

ecombinant neurolysin.

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