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Catalytic properties of thimet oligopeptidase H600A mutant
Biochemical and Biophysical Research Communications 394 (2010) 429–433
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Catalytic properties of thimet oligopeptidase H600A mutant Maurício F.M. Machado a, Marcelo F. Marcondes a, Vanessa Rioli b,c, Emer S. Ferro c, Maria A. Juliano a, Luiz Juliano a, Vitor Oliveira a,* a
Departamento de Biofísica, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil Laboratório Especial de Toxinologia Aplicada, Instituto Butantan, 05467-010 São Paulo, SP, Brazil c Departamento de Biologia Celular e Desenvolvimento, Universidade de São Paulo, 05508-900 São Paulo, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 1 March 2010 Available online 10 March 2010 Keywords: EP24.15 Substrate and inhibitor specificity Site-directed mutagenesis Thimet oligopeptidase
a b s t r a c t Thimet oligopeptidase (EC 3.4.24.15, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His600. In the present work, the role of His600 of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S1 and S01 specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His600 residue makes important interactions with the substrate, supporting the prediction that His600 moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both Km and kcat, showing the importance of His600 for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His600 in TOP catalysis, transferring a proton to the newly generated NH2-terminus or helping Tyr605 and/or Tyr612 in the intermediate oxyanion stabilization. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Thimet oligopeptidase (EC 3.4.24.15, TOP) is a 78 kDa Zn2+dependent endopeptidase, and member of M3 metallopeptidase family that contain the HEXXH motif [1]. TOP was initially detected and purified from soluble fraction of rat brain homogenates [2,3], is predominantly located in the cytosol of different cells. In vitro, TOP hydrolyses many bioactive peptides [4] and has been proposed to metabolize intracellular peptides, playing roles in antigen presentation [5–7]. TOP catalytic center is located in a deep channel that provides access only to short peptides [8]. This channel is lined with flexible loops [8] that allow the reorganization of the active site following substrate binding what is associated with the observed TOP broad substrate specificity [8–11]. TOP can accommodate different amino-acid residues at S4 to S03 [10], has high positive enthalpies and entropies for the activation indicating reorganization of the protein Abbreviations: TOP, thimet oligopeptidase; Dcp, E. coli dipeptidyl carboxypeptidase; ACE-2, angiotensin-converting enzyme (ACE)-related carboxypeptidase; MLN-4760, ((S,S)-2-(1-carboxy-2-(3-(3,5-dichlorobenzyl)-3himidazol4-ethylamino)-4-ethylpentanoic acid)). * Corresponding author at: Universidade Federal de São Paulo (UNIFESP), Department of Biophysics, Rua 3 de maio, 100 – Ed INFAR 2nd floor, São Paulo, Brazil. Fax: +55 11 55764450. E-mail address: [email protected] (V. Oliveira). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.03.045
structure and/or water during catalysis [12], it is activated by kosmotropic salt [12] and by the partial unfolding with urea [13]. The loop formed by residues 599–612 (G599HLAGGYDAQYYGY612), using TOP numbering, is in close proximity to its catalytic center. Site-directed mutagenesis studies in this loop showed the importance of Tyr612 and Tyr605 [14–16] for TOP hydrolytic activity. Recently, Bruce et al. [17] further evaluated the role of the Tyr605 and Tyr612 residues using double mutants (Y605/612F). The presence of the Gly residues was also investigated and showed to be important for the correct positioning of the two Tyr residues Tyr605 and Tyr612 [17]. In addition, these authors analyzed the data in the perspective of TOP structure models based on the observation that structurally related peptidases as Escherichia coli Dcp (dipeptidyl carboxypeptidase) and angiotensin-converting enzyme-related carboxypeptidase (ACE-2) undergo a large hinge movement upon substrate or inhibitor binding that causes their deep open channels to close around the substrate or inhibitor [18,19] suggesting that TOP may also present this characteristic. The analysis of the TOP structure based on the closed-forms of Dcp and ACE-2 indicates that some residues may move toward the substrate molecule during this hinge movement. Fig. 1 shows a possible relocation for His600 residue of TOP that corresponds to His601 in Dcp and His505 in ACE-2, which site-directed mutagenesis demonstrated that this residue is important for the catalysis of ACE-2 [20].
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Fig. 1. Structure fitting of TOP (1S4B), Dcp (1Y79) and ACE-2 (1R4L; closed form). Panel A – structure fitting of TOP (red) and ACE-2 (blue), showing the positions relative to the catalytic zinc ion (yellow sphere) of the residues His600 and Tyr605 of TOP and the counterparts His505 and Tyr510 of ACE-2. The black molecule represents the MLN-4760 inhibitor co-crystallized with ACE-2. Panel B – structure fitting of TOP (red) and Dcp (green), showing the positions relative to the catalytic zinc ion (yellow sphere) of the residues His600 and Tyr605 of TOP and the counterparts His601 and Tyr607 of Dcp. The black molecule represents the four residues (P2P1–P1´ P2´ ; Lys-Trp-Gly-Asp) of a hydrolyzed substrate present in the Dcp crystal structure. The figures were generated with the Swiss-pdb viewer v.4.0.1 software fixing the atoms from HEXXH motifs.
In the present work we examined the role of His600, from the loop G599HLAGGYDAQYYGY612 of TOP replacing this residue by an alanine residue, and then characterizing this TOP H600A mutant on its catalytic properties that include analysis of S1 and S01 specificity, pH-activity profile and inhibition by N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate (JA-2) a potent TOP inhibitor. 2. Materials and methods 2.1. Site-directed mutagenesis, protein expression, and purification The QuikChangeÒ site-directed mutagenesis kit (Stratagene) was used to introduce specific point mutations in TOP wild-type cDNAs, as described previously [7]. The point mutations were all confirmed by DNA sequencing [7] and identified as a TOP H600A. Wild-type and mutant proteins were expressed in E. coli DH5a using the pGEX4T-2 plasmid containing cDNA encoding the desired protein (GE Healthcare), as described previously [4]. The recombinant proteins were purified to homogeneity by affinity chromatography on a glutathione–Sepharose column (GE Healthcare). After purification, all of the proteins were analyzed using SDS/PAGE followed by staining with Coomassie blue [4]. Protein batches with a homogeneity >95% were stored at 80 °C and used in all subsequent analyses.
(automatic temperature compensation) glass probe. The reaction was monitored continuously based on the fluorescence of the released product. The rate of increase in fluorescence was converted into moles of substrate hydrolyzed based on the fluorescence curves of standard peptide solutions before and after total hydrolysis. The concentration of peptide solutions was determined by measuring the absorption of the 2,4-dinitrophenyl group at 365 nm (e = 17,300 M1 cm1). The enzyme concentration for initial rate determinations was chosen so that <5% of the substrate was hydrolyzed. The inner-filter effect was corrected using an empirical equation, and the kinetic parameters were calculated according to Wilkinson and using Eadie-Hofstee plots [14]. The data were analyzed and equations were fitted using Grafit (version 5.0; Erithacus Software). 2.4. Determination of inhibition parameters The TOP inhibitor JA-2 was kindly provided by Dr. A. Ian Smith (Monash University, Victoria, Australia). The inhibition was assayed using the FRET peptide Abz-GFSHFRQ-EDDnp as substrate. The Ki value was obtained from the equation Ki = Ki,app/(1 + [S]/ Km), and Ki,app was obtained using the equation Vo/Vi = 1 + [I]/Ki,app, where Vo = velocity of hydrolysis without the inhibitor, Vi = velocity of hydrolysis in the presence of the inhibitor, and [I] = molar concentration of the inhibitor.
2.2. Peptide synthesis 2.5. pH-Profile activities Two series of fluorescence resonance energy transfer (FRET) peptides derived from Abz-GFSPFRQ-EDDnp were synthesized by solid-phase procedures, as described previously [21]. In these peptides Q-EDDnp is the fluorescence acceptor and Abz is the fluorescence donor that correspond to glutamine-(N-(2,4-dinitrophenyl)ethylenediamine)) and ortho-aminobenzoic acid, respectively.
The kinetic parameters kcat/Km were determined over the pH range 5.0–9.8 using Abz-GFSAFRQ-EDDnp as the substrate in a four component buffer system of constant ionic strength, as describe above. DTT (0.5 mM) was added to assays using wild-type TOP and its mutants. Equations ((1) – shown below) were used in the non-linear regression analysis using the Grafit program.
2.3. Kinetic measurements
k¼ TOP activities were monitored spectrofluorimetrically in a Shimadzu RF-5301PC spectrofluorimeter using the FRET peptides as substrates, with excitation and emission wavelengths of 320 and 420 nm, respectively. A standard cuvette (1 cm path-length) containing 2 ml of substrate solution was placed in a thermostatically controlled cell compartment for 5 min before the addition of enzyme. Prior to the assay, TOP and its mutant was pre-activated by incubation with 0.5 mM DTT (dithiothrietol) for 5 min at 37 °C in 50 mM Tris/HCl buffer (pH 7.4), containing 100 mM NaCl. The pH was adjusted using a model 710 Orion pH meter with an ATC
Limit1 þ Limit2 10ðpHpK 1 Þ ðpHpK 1 Þ
10
þ1
Limit2 þ Limit3 10ðpHpK 2 Þ 10ðpHpK 2 Þ þ 1
ð1Þ
Eq. (1) fits data when the pH-activity profile depends upon two ionizing groups (double pKa) and does not assume that the activity is zero at high pH values. Limit1 represents the limit of the acid limb (low pH), Limit2 is the pH-independent maximum rate constant K1 and K2 are the dissociation constants of a catalytically competent base and acids, respectively, and Limit3 is the limit of the alkaline limb (high pH), k = kcat/Km. In this work, Eq. (1) was used to study the pH–kcat/Km profile of wild-type TOP and mutants H600A, otherwise Eq. (2) was used.
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M.F.M. Machado et al. / Biochemical and Biophysical Research Communications 394 (2010) 429–433 Table 1 Kinetic parameters for the hydrolysis of FRET peptides series Abz-GFSPXRQ-EDDnp and Abz-GFSPXRQ-EDDnp by TOP wild-type and mutant H600A. X
A I F L W S Q E D R H P
Abz-GFa;SP;XRQ-EDDnp
Abz-GFSX;FRQ-EDDnp 1
Km (lM)
kcat (s
14.3 (1.2) 112.0 (0.4) 10.5 (0.7) – 20.8 (0.8) 12.7 (3.7) 9.1(1.2) 1.1 (7.5) 0.9 (3.8) 10.9(1.1) 32.1 (7.6) 9.3 (2.2)
11.1 (10.2) 0.19 (0.01) 5.9 (6.7) – 6.0 (2.7) 9.2 (8.8) 7.0 (6.3) 1.6 (1.6) 0.3 (0.7) 9.1 (7.0) 13.3 (24.7) 2.9 (6.3)
)
kcat/Km (s
1
1
mM
)
1600 (8500) 2 (25) 561 (9571) – 288 (3375) 724 (2400) 771 (5250) 1417 (200) 333 (200) 839 (6363) 414 (3300) 317 (2863)
Km (lM)
kcat (s1)
kcat/Km (s1 mM1)
21.8 (4.0) 6.2 (8.5) 9.3 (2.2) 12.2 (4.5) – 13.5 (7.3) 14.8 (4.3) 19.2 (18.0) 7.5 (37.0) 21.3 (1.8) 13.1 (1.5) –
1.7 (18.5) 0.04 (5.9) 2.9 (6.3) a 0.02 (0.74) – 0.4 (21.5) 0.7 (12.3) a 0.01 (1.8) a 0.03 (8.2) 4.5 (2.7) 0.3 (5.3) –
78 (4600) 10 (1100) 317 (2863) 8 (1100) – 30 (2900) 47 (2900) 1.0 (200) 8.0 (500) 211 (1500) 20.6 (3500) –
a
The arrows indicate the cleavage site in each series. a These peptides were cleaved by TOP wild-type and TOP H600A mutant at the peptide bonds P–X and F–S with almost similar preference. The values in parentheses are for TOP wild-type as earlier reported [7]. The data are presented as means ± SD, with SD (not shown) being <10% of the values indicated.
k ¼ kðLimitÞ½1=1 þ 10ðpK 1 pHÞ þ 10ðpHpK 2 Þ
ð2Þ
kcat /KM mutant/ kcat /KM wild type (log scale)
kcat /KM mutant/ kcat /KM wild type (log scale)
Eq. (2) fits data when the pH-activity profile also depends upon two ionizing groups in a bell-shaped curve and the activities at low and high pHs are zero. k(Limit) corresponds to the pH-independent maximum rate constant, K1 and K2 are the dissociation constants of a catalytically competent base and acid, respectively, and k = kcat or kcat/Km. Eq. (2) was used in the non-linear regression analysis of pH–kcat of wild-type TOP and mutants H600A The pK1 and pK2 estimated from the pH–kcat/Km curves were identified as pKe1 and pKe2, respectively, to differentiate them from the pK1 and pK2 values estimated from the pH–kcat profiles (pKes1 and pKes2, respectively).
3. Results 3.1. Kinetic parameters of hydrolysis by TOP H600A mutant of FRET peptides substrates modified at the P1 and P01 positions The kinetic parameters for hydrolysis by TOP H600A mutant of the FRET peptide series Abz-GFSXFRQ-EDDnp (X = I, F, W, S, Q, E, D, R, H, and P) and Abz-GFSPXRQ-EDDnp (X = A, I, L, S, Q, D, E, N, H, R, and F) are showed in Table 1 and the comparison of catalytic efficiency (kcat/Km) values between TOP wild-type and H600A mutant is shown in Fig. 2. These two series of peptides are derived from
10
1
0.1
0.01 A
I
F
W
S
Q P1
E
D
R
H
P
A
I
L
S
Q
D P1'
E
N
H
R
F
1
0.1
0.01
0.001
Fig. 2. Relative kcat/Km ratios for the of TOP hydrolysis of peptides derived from (A) Abz-GFSXFRQ-EDDnp and (B) Abz-GFSPXRQ-EDDnp by wild-type and H600A mutant. The log scale allows rapid comparison of ratios greater or less than 1.
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Abz-GFSPFRQ-EDDnp that contains the six C-terminal amino acids of bradykinin that is an efficient substrate for TOP [10]. All the peptides from the series Abz-GFSXFRQ-EDDnp were cleaved at the X;F peptide bond and those of Abz-GFSPXRQ-EDDnp were all cleaved at P;X except the peptides with X = E, D, I, and L that presented an additional cleavage at F;S bond. TOP H600A mutant presented lower catalytic efficiencies than the wild-type for the hydrolysis of most peptides of both series Abz-GFSX;FRQ-EDDnp and Abz-GFSP;XRQ-EDDnp. It is noteworthy that in the series Abz-GFSX;FRQ-EDDnp, the similarity of the kcat values obtained for the hydrolysis of the substrates by TOP wild-type and H600A mutant, while the Km values were significantly lower with TOP wild-type. The exceptions were the substrates containing the negatively charged residues E and D that in the P1 position of the series Abz-GFSX;FRQ-EDDnp were hydrolyzed by TOP H600A mutant with higher kcat/Km values compared to TOP wild-type. Furthermore, these differences in kcat/Km values were due to the Km values that were lower for the hydrolysis of Abz-GFSD;FRQ-EDDnp and Abz-GFSE;FRQ-EDDnp by TOP H600A mutant. Although Km does not exclusively represent the affinity of enzyme–substrate complex, it is reasonable to accept that His600 plays significant role on the binding of the substrates in the TOP catalytic center. The largest effect of the mutation H600A were observed in the kinetic parameters for the hydrolysis of the substrates in the series Abz-GFSPXRQ-EDDnp with variations at P01 position. These substrates were hydrolyzed with kcat/ Km ratios approximately 100-fold lower by the TOP H600A mutant in comparison with the wild-type enzyme, with exception of the substrates with Arg and Phe. In addition, these differences were not only due to alterations in Km like the results obtained with the substrates containing changes at P1 (Table 1; Fig. 2) but, also due to much lower kcat constants obtained with the H600A mutant for these substrates containing changes at P01 specially the substrates containing Asp, Glu, or Ile.
Fig. 3. pH-Profiles of TOP wild-type and H600A mutant activities. pH Dependence of (A) kcat/Km for wild-type TOP (open circles) and TOP H600A (full circles), and (B) kcat for the hydolysis of Abz-GFSAFRQ-EDDnp by wild-type TOP (open circles) and TOP H600A (full circles). Solid lines represent the curves calculated as indicated in Section 2.
3.2. Inhibition by JA-2 The Ki values for the inhibition of TOP wild-type and H600A mutant by the JA-2 compound were 45 nM and 33 nM, respectively. This data shows that residue His600 does not contribute significantly to the JA-2 binding.
of more than one ionic group, and thus the associated pKs are essentially macroscopic constants [14,16].
3.3. pH-Profile activities of TOP H600A mutant and wild-type
The modification of residue 600 in TOP H600A significantly affected the kinetic parameters for the hydrolysis by this mutant in comparison with the wild-type TOP (Tables 1 and 2). The mutant TOP H600A presented lower catalytic efficiencies than the wildtype in the kinetic assays with all peptides of the series AbzGFSPXRQ-EDDnp and also the peptides of the series AbzGFSXFRQ-EDDnp with exception of the substrates containing the negatively charged residues Glu and Asp at the P1 position. Interestingly these two peptides were hydrolyzed more efficiently by the TOP H600A mutant than by the TOP wild-type (Table 1 and Fig. 3) mainly because these two peptides presented higher affinities by this TOP mutant than by the wild-type peptidase. These differences in the catalytic efficiencies of TOP H600A and TOP wildtype according with the residue at P1 may indicate that the His600 is important for the recognition of the amino acid of the substrate at this position. However, this difference is only significant in the substrates containing with these two negatively charged residues at P1. The prediction based on the Dcp and ACE-2 structures point to a direct interaction between the His600 and the substrate main chain by a hydrogen bond between the His Ne and the carbonyl oxygen from the P01 –P02 bond, and the imidazole ring would interact with the P1 side chain depending on the residue at this substrate position (Fig. 1) [18]. Furthermore, the largest effects of the mutation H600A were observed in the kinetic assays with
Table 2 shows pKe1, pKe2, pKes1, and pKes2 values calculated from the profiles of pH versus kcat/Km values and pH versus kcat values of TOP wild-type and H600A mutant. The replacement of the His600 by an alanine residue did not altered the acid limb of the pH– kcat/Km and pH–kcat profiles, and thus no significant differences between the pKe1 and pKes1 values were observed (Fig. 3A and Table 2). On the other hand, it is clear the effect of the His600 residue to the alkaline limb of both profiles, resulting that the pKe2 and pKes2 are significantly different for the enzymes. These results support previous pH studies and the interpretation that the alkaline limb of TOP activity pH-profiles involves the contribution
Table 2 Kinetic parameters for the hydrolysis of Abz-GFSAFRQ-EDDnp by TOP wild-type and mutant H600A calculated from the pH-rate curves. kcat/Km
WT H600A *
kcat
pKe1
pKe2
pKes1
pKes2
6.2 ± 0.2* 6.1 ± 0.1
8.1 ± 0.1* 7.4 ± 0.1
5.4 ± 0.2* 5.7 ± 0.2
9.3 ± 0.2* 8.4 ± 0.1
Indicates data from Ref. [14].
4. Discussions
M.F.M. Machado et al. / Biochemical and Biophysical Research Communications 394 (2010) 429–433
the substrates of the series Abz-GFSPXRQ-EDDnp. These substrates were hydrolyzed with kcat/Km ratios approximately 100-fold lower by the TOP H600A mutant in comparison with the wild-type enzyme, with exception of the substrates with Arg and Phe at this position. In addition, these differences were not only due to alterations in Km like the results obtained with the substrates containing changes at P1 (Table 1; Fig. 3) but, also due to much lower kcat constants obtained with the H600A mutant for these substrates containing changes at P01 specially the substrates containing Asp, Glu or Ile (285-, 165-, and 69-fold). The differences observed in the pH-profiles of TOP wild-type and mutant H600A show that the His600 residue is important for both substrate binding and catalysis but, the higher difference in the pKes2 constants (pH–kcat dependence curves) may imply a major contribution of this residue for one or more steps that affects the catalytic constants of TOP catalyzed reactions. His600 could be responsible for the proton transfer to the newly generated NH2terminus, or it could be possible that His600 helps Tyr605 and/or Tyr612 [14,15,17] in the stabilization of the oxyanion formed in the tetrahedral intermediate during catalysis. The latter possibility is proposed for the counterpart His505 residue in ACE-2 [20]. Similar co participation of histidine and tyrosine residues in the oxyanion stabilization is present in thermolysin [22]. Further studies particularly with prototype metallopeptidases could help to better define the auxiliary residues in the catalytic machinery of this class of peptidases. 5. Conclusion Taken together, the data presented here support the prediction that His600 residue of TOP move towards to the substrate due to a hinge movement that occurs during TOP catalysis, similar to Dcp and ACE-2 (Fig. 1). The results with the substrates containing different amino-acid residues at P1 and P01 positions and the results of the pH assays show that His600 of TOP makes important interactions with the substrate, and that such interactions take place on both substrate binding and the product release from TOP active site. Acknowledgments This work was supported by Grants from Sao Paulo State Research Foundation (FAPESP) MFMM (08/57336-2). V.O. is supported by research fellowship from CNPq. References [1] A. Pierotti, K.W. Dong, M.J. Glucksman, M. Orlowski, J.L. Roberts, Molecular cloning and primary structure of rat testes metalloendopeptidase EC 3.4.24.15, Biochemistry 29 (1990) 10323–10329. [2] M. Orlowski, C. Michaud, T.G. Chu, A soluble metalloendopeptidase from rat brain. Purification of the enzyme and determination of specificity with synthetic and natural peptides, Eur. J. Biochem. 135 (1983) 81–88. [3] F. Checler, J.P. Vincent, P. Kitabgi, Purification and characterization of a novel neurotensin-degrading peptidase from rat brain synaptic membranes, J. Biol. Chem. 261 (1986) 11274–11281.
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Catalytic properties of thimet oligopeptidase H600A mutant Maurício F.M. Machado a, Marcelo F. Marcondes a, Vanessa Rioli b,c, Emer S. Ferro c, Maria A. Juliano a, Luiz Juliano a, Vitor Oliveira a,* a
Departamento de Biofísica, Universidade Federal de São Paulo, 04044-020 São Paulo, SP, Brazil Laboratório Especial de Toxinologia Aplicada, Instituto Butantan, 05467-010 São Paulo, SP, Brazil c Departamento de Biologia Celular e Desenvolvimento, Universidade de São Paulo, 05508-900 São Paulo, SP, Brazil b
a r t i c l e
i n f o
Article history: Received 1 March 2010 Available online 10 March 2010 Keywords: EP24.15 Substrate and inhibitor specificity Site-directed mutagenesis Thimet oligopeptidase
a b s t r a c t Thimet oligopeptidase (EC 3.4.24.15, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His600. In the present work, the role of His600 of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S1 and S01 specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His600 residue makes important interactions with the substrate, supporting the prediction that His600 moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both Km and kcat, showing the importance of His600 for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His600 in TOP catalysis, transferring a proton to the newly generated NH2-terminus or helping Tyr605 and/or Tyr612 in the intermediate oxyanion stabilization. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Thimet oligopeptidase (EC 3.4.24.15, TOP) is a 78 kDa Zn2+dependent endopeptidase, and member of M3 metallopeptidase family that contain the HEXXH motif [1]. TOP was initially detected and purified from soluble fraction of rat brain homogenates [2,3], is predominantly located in the cytosol of different cells. In vitro, TOP hydrolyses many bioactive peptides [4] and has been proposed to metabolize intracellular peptides, playing roles in antigen presentation [5–7]. TOP catalytic center is located in a deep channel that provides access only to short peptides [8]. This channel is lined with flexible loops [8] that allow the reorganization of the active site following substrate binding what is associated with the observed TOP broad substrate specificity [8–11]. TOP can accommodate different amino-acid residues at S4 to S03 [10], has high positive enthalpies and entropies for the activation indicating reorganization of the protein Abbreviations: TOP, thimet oligopeptidase; Dcp, E. coli dipeptidyl carboxypeptidase; ACE-2, angiotensin-converting enzyme (ACE)-related carboxypeptidase; MLN-4760, ((S,S)-2-(1-carboxy-2-(3-(3,5-dichlorobenzyl)-3himidazol4-ethylamino)-4-ethylpentanoic acid)). * Corresponding author at: Universidade Federal de São Paulo (UNIFESP), Department of Biophysics, Rua 3 de maio, 100 – Ed INFAR 2nd floor, São Paulo, Brazil. Fax: +55 11 55764450. E-mail address: [email protected] (V. Oliveira). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.03.045
structure and/or water during catalysis [12], it is activated by kosmotropic salt [12] and by the partial unfolding with urea [13]. The loop formed by residues 599–612 (G599HLAGGYDAQYYGY612), using TOP numbering, is in close proximity to its catalytic center. Site-directed mutagenesis studies in this loop showed the importance of Tyr612 and Tyr605 [14–16] for TOP hydrolytic activity. Recently, Bruce et al. [17] further evaluated the role of the Tyr605 and Tyr612 residues using double mutants (Y605/612F). The presence of the Gly residues was also investigated and showed to be important for the correct positioning of the two Tyr residues Tyr605 and Tyr612 [17]. In addition, these authors analyzed the data in the perspective of TOP structure models based on the observation that structurally related peptidases as Escherichia coli Dcp (dipeptidyl carboxypeptidase) and angiotensin-converting enzyme-related carboxypeptidase (ACE-2) undergo a large hinge movement upon substrate or inhibitor binding that causes their deep open channels to close around the substrate or inhibitor [18,19] suggesting that TOP may also present this characteristic. The analysis of the TOP structure based on the closed-forms of Dcp and ACE-2 indicates that some residues may move toward the substrate molecule during this hinge movement. Fig. 1 shows a possible relocation for His600 residue of TOP that corresponds to His601 in Dcp and His505 in ACE-2, which site-directed mutagenesis demonstrated that this residue is important for the catalysis of ACE-2 [20].
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Fig. 1. Structure fitting of TOP (1S4B), Dcp (1Y79) and ACE-2 (1R4L; closed form). Panel A – structure fitting of TOP (red) and ACE-2 (blue), showing the positions relative to the catalytic zinc ion (yellow sphere) of the residues His600 and Tyr605 of TOP and the counterparts His505 and Tyr510 of ACE-2. The black molecule represents the MLN-4760 inhibitor co-crystallized with ACE-2. Panel B – structure fitting of TOP (red) and Dcp (green), showing the positions relative to the catalytic zinc ion (yellow sphere) of the residues His600 and Tyr605 of TOP and the counterparts His601 and Tyr607 of Dcp. The black molecule represents the four residues (P2P1–P1´ P2´ ; Lys-Trp-Gly-Asp) of a hydrolyzed substrate present in the Dcp crystal structure. The figures were generated with the Swiss-pdb viewer v.4.0.1 software fixing the atoms from HEXXH motifs.
In the present work we examined the role of His600, from the loop G599HLAGGYDAQYYGY612 of TOP replacing this residue by an alanine residue, and then characterizing this TOP H600A mutant on its catalytic properties that include analysis of S1 and S01 specificity, pH-activity profile and inhibition by N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate (JA-2) a potent TOP inhibitor. 2. Materials and methods 2.1. Site-directed mutagenesis, protein expression, and purification The QuikChangeÒ site-directed mutagenesis kit (Stratagene) was used to introduce specific point mutations in TOP wild-type cDNAs, as described previously [7]. The point mutations were all confirmed by DNA sequencing [7] and identified as a TOP H600A. Wild-type and mutant proteins were expressed in E. coli DH5a using the pGEX4T-2 plasmid containing cDNA encoding the desired protein (GE Healthcare), as described previously [4]. The recombinant proteins were purified to homogeneity by affinity chromatography on a glutathione–Sepharose column (GE Healthcare). After purification, all of the proteins were analyzed using SDS/PAGE followed by staining with Coomassie blue [4]. Protein batches with a homogeneity >95% were stored at 80 °C and used in all subsequent analyses.
(automatic temperature compensation) glass probe. The reaction was monitored continuously based on the fluorescence of the released product. The rate of increase in fluorescence was converted into moles of substrate hydrolyzed based on the fluorescence curves of standard peptide solutions before and after total hydrolysis. The concentration of peptide solutions was determined by measuring the absorption of the 2,4-dinitrophenyl group at 365 nm (e = 17,300 M1 cm1). The enzyme concentration for initial rate determinations was chosen so that <5% of the substrate was hydrolyzed. The inner-filter effect was corrected using an empirical equation, and the kinetic parameters were calculated according to Wilkinson and using Eadie-Hofstee plots [14]. The data were analyzed and equations were fitted using Grafit (version 5.0; Erithacus Software). 2.4. Determination of inhibition parameters The TOP inhibitor JA-2 was kindly provided by Dr. A. Ian Smith (Monash University, Victoria, Australia). The inhibition was assayed using the FRET peptide Abz-GFSHFRQ-EDDnp as substrate. The Ki value was obtained from the equation Ki = Ki,app/(1 + [S]/ Km), and Ki,app was obtained using the equation Vo/Vi = 1 + [I]/Ki,app, where Vo = velocity of hydrolysis without the inhibitor, Vi = velocity of hydrolysis in the presence of the inhibitor, and [I] = molar concentration of the inhibitor.
2.2. Peptide synthesis 2.5. pH-Profile activities Two series of fluorescence resonance energy transfer (FRET) peptides derived from Abz-GFSPFRQ-EDDnp were synthesized by solid-phase procedures, as described previously [21]. In these peptides Q-EDDnp is the fluorescence acceptor and Abz is the fluorescence donor that correspond to glutamine-(N-(2,4-dinitrophenyl)ethylenediamine)) and ortho-aminobenzoic acid, respectively.
The kinetic parameters kcat/Km were determined over the pH range 5.0–9.8 using Abz-GFSAFRQ-EDDnp as the substrate in a four component buffer system of constant ionic strength, as describe above. DTT (0.5 mM) was added to assays using wild-type TOP and its mutants. Equations ((1) – shown below) were used in the non-linear regression analysis using the Grafit program.
2.3. Kinetic measurements
k¼ TOP activities were monitored spectrofluorimetrically in a Shimadzu RF-5301PC spectrofluorimeter using the FRET peptides as substrates, with excitation and emission wavelengths of 320 and 420 nm, respectively. A standard cuvette (1 cm path-length) containing 2 ml of substrate solution was placed in a thermostatically controlled cell compartment for 5 min before the addition of enzyme. Prior to the assay, TOP and its mutant was pre-activated by incubation with 0.5 mM DTT (dithiothrietol) for 5 min at 37 °C in 50 mM Tris/HCl buffer (pH 7.4), containing 100 mM NaCl. The pH was adjusted using a model 710 Orion pH meter with an ATC
Limit1 þ Limit2 10ðpHpK 1 Þ ðpHpK 1 Þ
10
þ1
Limit2 þ Limit3 10ðpHpK 2 Þ 10ðpHpK 2 Þ þ 1
ð1Þ
Eq. (1) fits data when the pH-activity profile depends upon two ionizing groups (double pKa) and does not assume that the activity is zero at high pH values. Limit1 represents the limit of the acid limb (low pH), Limit2 is the pH-independent maximum rate constant K1 and K2 are the dissociation constants of a catalytically competent base and acids, respectively, and Limit3 is the limit of the alkaline limb (high pH), k = kcat/Km. In this work, Eq. (1) was used to study the pH–kcat/Km profile of wild-type TOP and mutants H600A, otherwise Eq. (2) was used.
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M.F.M. Machado et al. / Biochemical and Biophysical Research Communications 394 (2010) 429–433 Table 1 Kinetic parameters for the hydrolysis of FRET peptides series Abz-GFSPXRQ-EDDnp and Abz-GFSPXRQ-EDDnp by TOP wild-type and mutant H600A. X
A I F L W S Q E D R H P
Abz-GFa;SP;XRQ-EDDnp
Abz-GFSX;FRQ-EDDnp 1
Km (lM)
kcat (s
14.3 (1.2) 112.0 (0.4) 10.5 (0.7) – 20.8 (0.8) 12.7 (3.7) 9.1(1.2) 1.1 (7.5) 0.9 (3.8) 10.9(1.1) 32.1 (7.6) 9.3 (2.2)
11.1 (10.2) 0.19 (0.01) 5.9 (6.7) – 6.0 (2.7) 9.2 (8.8) 7.0 (6.3) 1.6 (1.6) 0.3 (0.7) 9.1 (7.0) 13.3 (24.7) 2.9 (6.3)
)
kcat/Km (s
1
1
mM
)
1600 (8500) 2 (25) 561 (9571) – 288 (3375) 724 (2400) 771 (5250) 1417 (200) 333 (200) 839 (6363) 414 (3300) 317 (2863)
Km (lM)
kcat (s1)
kcat/Km (s1 mM1)
21.8 (4.0) 6.2 (8.5) 9.3 (2.2) 12.2 (4.5) – 13.5 (7.3) 14.8 (4.3) 19.2 (18.0) 7.5 (37.0) 21.3 (1.8) 13.1 (1.5) –
1.7 (18.5) 0.04 (5.9) 2.9 (6.3) a 0.02 (0.74) – 0.4 (21.5) 0.7 (12.3) a 0.01 (1.8) a 0.03 (8.2) 4.5 (2.7) 0.3 (5.3) –
78 (4600) 10 (1100) 317 (2863) 8 (1100) – 30 (2900) 47 (2900) 1.0 (200) 8.0 (500) 211 (1500) 20.6 (3500) –
a
The arrows indicate the cleavage site in each series. a These peptides were cleaved by TOP wild-type and TOP H600A mutant at the peptide bonds P–X and F–S with almost similar preference. The values in parentheses are for TOP wild-type as earlier reported [7]. The data are presented as means ± SD, with SD (not shown) being <10% of the values indicated.
k ¼ kðLimitÞ½1=1 þ 10ðpK 1 pHÞ þ 10ðpHpK 2 Þ
ð2Þ
kcat /KM mutant/ kcat /KM wild type (log scale)
kcat /KM mutant/ kcat /KM wild type (log scale)
Eq. (2) fits data when the pH-activity profile also depends upon two ionizing groups in a bell-shaped curve and the activities at low and high pHs are zero. k(Limit) corresponds to the pH-independent maximum rate constant, K1 and K2 are the dissociation constants of a catalytically competent base and acid, respectively, and k = kcat or kcat/Km. Eq. (2) was used in the non-linear regression analysis of pH–kcat of wild-type TOP and mutants H600A The pK1 and pK2 estimated from the pH–kcat/Km curves were identified as pKe1 and pKe2, respectively, to differentiate them from the pK1 and pK2 values estimated from the pH–kcat profiles (pKes1 and pKes2, respectively).
3. Results 3.1. Kinetic parameters of hydrolysis by TOP H600A mutant of FRET peptides substrates modified at the P1 and P01 positions The kinetic parameters for hydrolysis by TOP H600A mutant of the FRET peptide series Abz-GFSXFRQ-EDDnp (X = I, F, W, S, Q, E, D, R, H, and P) and Abz-GFSPXRQ-EDDnp (X = A, I, L, S, Q, D, E, N, H, R, and F) are showed in Table 1 and the comparison of catalytic efficiency (kcat/Km) values between TOP wild-type and H600A mutant is shown in Fig. 2. These two series of peptides are derived from
10
1
0.1
0.01 A
I
F
W
S
Q P1
E
D
R
H
P
A
I
L
S
Q
D P1'
E
N
H
R
F
1
0.1
0.01
0.001
Fig. 2. Relative kcat/Km ratios for the of TOP hydrolysis of peptides derived from (A) Abz-GFSXFRQ-EDDnp and (B) Abz-GFSPXRQ-EDDnp by wild-type and H600A mutant. The log scale allows rapid comparison of ratios greater or less than 1.
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Abz-GFSPFRQ-EDDnp that contains the six C-terminal amino acids of bradykinin that is an efficient substrate for TOP [10]. All the peptides from the series Abz-GFSXFRQ-EDDnp were cleaved at the X;F peptide bond and those of Abz-GFSPXRQ-EDDnp were all cleaved at P;X except the peptides with X = E, D, I, and L that presented an additional cleavage at F;S bond. TOP H600A mutant presented lower catalytic efficiencies than the wild-type for the hydrolysis of most peptides of both series Abz-GFSX;FRQ-EDDnp and Abz-GFSP;XRQ-EDDnp. It is noteworthy that in the series Abz-GFSX;FRQ-EDDnp, the similarity of the kcat values obtained for the hydrolysis of the substrates by TOP wild-type and H600A mutant, while the Km values were significantly lower with TOP wild-type. The exceptions were the substrates containing the negatively charged residues E and D that in the P1 position of the series Abz-GFSX;FRQ-EDDnp were hydrolyzed by TOP H600A mutant with higher kcat/Km values compared to TOP wild-type. Furthermore, these differences in kcat/Km values were due to the Km values that were lower for the hydrolysis of Abz-GFSD;FRQ-EDDnp and Abz-GFSE;FRQ-EDDnp by TOP H600A mutant. Although Km does not exclusively represent the affinity of enzyme–substrate complex, it is reasonable to accept that His600 plays significant role on the binding of the substrates in the TOP catalytic center. The largest effect of the mutation H600A were observed in the kinetic parameters for the hydrolysis of the substrates in the series Abz-GFSPXRQ-EDDnp with variations at P01 position. These substrates were hydrolyzed with kcat/ Km ratios approximately 100-fold lower by the TOP H600A mutant in comparison with the wild-type enzyme, with exception of the substrates with Arg and Phe. In addition, these differences were not only due to alterations in Km like the results obtained with the substrates containing changes at P1 (Table 1; Fig. 2) but, also due to much lower kcat constants obtained with the H600A mutant for these substrates containing changes at P01 specially the substrates containing Asp, Glu, or Ile.
Fig. 3. pH-Profiles of TOP wild-type and H600A mutant activities. pH Dependence of (A) kcat/Km for wild-type TOP (open circles) and TOP H600A (full circles), and (B) kcat for the hydolysis of Abz-GFSAFRQ-EDDnp by wild-type TOP (open circles) and TOP H600A (full circles). Solid lines represent the curves calculated as indicated in Section 2.
3.2. Inhibition by JA-2 The Ki values for the inhibition of TOP wild-type and H600A mutant by the JA-2 compound were 45 nM and 33 nM, respectively. This data shows that residue His600 does not contribute significantly to the JA-2 binding.
of more than one ionic group, and thus the associated pKs are essentially macroscopic constants [14,16].
3.3. pH-Profile activities of TOP H600A mutant and wild-type
The modification of residue 600 in TOP H600A significantly affected the kinetic parameters for the hydrolysis by this mutant in comparison with the wild-type TOP (Tables 1 and 2). The mutant TOP H600A presented lower catalytic efficiencies than the wildtype in the kinetic assays with all peptides of the series AbzGFSPXRQ-EDDnp and also the peptides of the series AbzGFSXFRQ-EDDnp with exception of the substrates containing the negatively charged residues Glu and Asp at the P1 position. Interestingly these two peptides were hydrolyzed more efficiently by the TOP H600A mutant than by the TOP wild-type (Table 1 and Fig. 3) mainly because these two peptides presented higher affinities by this TOP mutant than by the wild-type peptidase. These differences in the catalytic efficiencies of TOP H600A and TOP wildtype according with the residue at P1 may indicate that the His600 is important for the recognition of the amino acid of the substrate at this position. However, this difference is only significant in the substrates containing with these two negatively charged residues at P1. The prediction based on the Dcp and ACE-2 structures point to a direct interaction between the His600 and the substrate main chain by a hydrogen bond between the His Ne and the carbonyl oxygen from the P01 –P02 bond, and the imidazole ring would interact with the P1 side chain depending on the residue at this substrate position (Fig. 1) [18]. Furthermore, the largest effects of the mutation H600A were observed in the kinetic assays with
Table 2 shows pKe1, pKe2, pKes1, and pKes2 values calculated from the profiles of pH versus kcat/Km values and pH versus kcat values of TOP wild-type and H600A mutant. The replacement of the His600 by an alanine residue did not altered the acid limb of the pH– kcat/Km and pH–kcat profiles, and thus no significant differences between the pKe1 and pKes1 values were observed (Fig. 3A and Table 2). On the other hand, it is clear the effect of the His600 residue to the alkaline limb of both profiles, resulting that the pKe2 and pKes2 are significantly different for the enzymes. These results support previous pH studies and the interpretation that the alkaline limb of TOP activity pH-profiles involves the contribution
Table 2 Kinetic parameters for the hydrolysis of Abz-GFSAFRQ-EDDnp by TOP wild-type and mutant H600A calculated from the pH-rate curves. kcat/Km
WT H600A *
kcat
pKe1
pKe2
pKes1
pKes2
6.2 ± 0.2* 6.1 ± 0.1
8.1 ± 0.1* 7.4 ± 0.1
5.4 ± 0.2* 5.7 ± 0.2
9.3 ± 0.2* 8.4 ± 0.1
Indicates data from Ref. [14].
4. Discussions
M.F.M. Machado et al. / Biochemical and Biophysical Research Communications 394 (2010) 429–433
the substrates of the series Abz-GFSPXRQ-EDDnp. These substrates were hydrolyzed with kcat/Km ratios approximately 100-fold lower by the TOP H600A mutant in comparison with the wild-type enzyme, with exception of the substrates with Arg and Phe at this position. In addition, these differences were not only due to alterations in Km like the results obtained with the substrates containing changes at P1 (Table 1; Fig. 3) but, also due to much lower kcat constants obtained with the H600A mutant for these substrates containing changes at P01 specially the substrates containing Asp, Glu or Ile (285-, 165-, and 69-fold). The differences observed in the pH-profiles of TOP wild-type and mutant H600A show that the His600 residue is important for both substrate binding and catalysis but, the higher difference in the pKes2 constants (pH–kcat dependence curves) may imply a major contribution of this residue for one or more steps that affects the catalytic constants of TOP catalyzed reactions. His600 could be responsible for the proton transfer to the newly generated NH2terminus, or it could be possible that His600 helps Tyr605 and/or Tyr612 [14,15,17] in the stabilization of the oxyanion formed in the tetrahedral intermediate during catalysis. The latter possibility is proposed for the counterpart His505 residue in ACE-2 [20]. Similar co participation of histidine and tyrosine residues in the oxyanion stabilization is present in thermolysin [22]. Further studies particularly with prototype metallopeptidases could help to better define the auxiliary residues in the catalytic machinery of this class of peptidases. 5. Conclusion Taken together, the data presented here support the prediction that His600 residue of TOP move towards to the substrate due to a hinge movement that occurs during TOP catalysis, similar to Dcp and ACE-2 (Fig. 1). The results with the substrates containing different amino-acid residues at P1 and P01 positions and the results of the pH assays show that His600 of TOP makes important interactions with the substrate, and that such interactions take place on both substrate binding and the product release from TOP active site. Acknowledgments This work was supported by Grants from Sao Paulo State Research Foundation (FAPESP) MFMM (08/57336-2). V.O. is supported by research fellowship from CNPq. References [1] A. Pierotti, K.W. Dong, M.J. Glucksman, M. Orlowski, J.L. Roberts, Molecular cloning and primary structure of rat testes metalloendopeptidase EC 3.4.24.15, Biochemistry 29 (1990) 10323–10329. [2] M. Orlowski, C. Michaud, T.G. Chu, A soluble metalloendopeptidase from rat brain. Purification of the enzyme and determination of specificity with synthetic and natural peptides, Eur. J. Biochem. 135 (1983) 81–88. [3] F. Checler, J.P. Vincent, P. Kitabgi, Purification and characterization of a novel neurotensin-degrading peptidase from rat brain synaptic membranes, J. Biol. Chem. 261 (1986) 11274–11281.
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