Active Site of Deblocking Aminopeptidase from Pyrococcus horikoshii

Biochemical and Biophysical Research Communications 290, 994 –997 (2002) doi:10.1006/bbrc.2001.6327, available online at http://www.idealibrary.com on

Active Site of Deblocking Aminopeptidase from Pyrococcus horikoshii Shinji Onoe,* Susumu Ando,* ,† Mitsuo Ataka,* and Kazuhiko Ishikawa* ,1 *National Institute of Advanced Industrial Science and Technology (Kansai), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan; and †Rakuto Kasei Industrial Company Inc., 5-1 Sekinotsu 4-Chome, Otsu, Shiga 520-2277, Japan

Received December 25, 2001

New hyperthermostable aminopeptidase from the hyperthermophilic archaeon Pyrococcus horikoshii has acylamino acid releasing (deblocking) activity for acyl (blocked) peptides. Such an enzyme can be used for N-terminal sequencing of acyl peptides. To clarify the active site of the deblocking aminopeptidase, we prepared three mutants in which one of the three possible active site amino acid residues (Asp or Glu) was replaced with their amide derivatives. Activity and cobalt ion dependence of these mutants were examined and compared with those of the native enzyme. The results suggest that all the three possible residues (Asp173, Glu205, and Glu206) participate in the catalytic activity through binding with the cobalt ion. © 2002 Elsevier Science (USA) Key Words: thermostable; hyperthermostable; archaeon; cobalt; acyl peptide; mutant.

Pyrococcus horikoshii OT3 is one of the thermophilic archaea that has optimal growth temperature at 98°C (1). Most of the proteins from P. horikoshii are thought to be hyperthermostable (2). A new thermostable aminopeptidase was found from P. horikoshii (3). This enzyme is a novel aminopeptidase that has acylamino acid releasing activity. An enzyme with such activity was first found by Tsunasawa et al. in P. furiosus and named deblocking aminopeptidase (DAP) (4). It is already used commercially for N-terminal sequencing of acyl peptides (4, 5), but the characteristics of the enzyme have not been reported. The activity of deblocking has not been found in the normal aminopeptidases. In case of normal aminopeptidase from Aeromonas proteolytica (hereafter referred to as APR), the crystallographic analysis showed that 1

To whom correspondence should be addressed at The Special Division for Human Life Technology, National Institute of Advanced Industrial Science and Technology (Kansai), 1-8-31, Midorigaoka, Ikeda, Osaka 563-8577, Japan. Fax: 81-727-51-9628. E-mail: [email protected]. 0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

the Asp117, Glu151 and Glu152 constitute a cocatalytic unit that can bind two zinc ions (6, 7). The homology between APR and DAP was not observed except for the two regions shown in the boxes in Fig. 1. We have presently no information about the structure and active site of DAP. However, Asp173, Glu205, and Glu206 of the DAP from P. horikoshii (hereafter referred to as DAPPh), located in the highly conserved regions, correspond to the Asp117, Glu151, and Glu152 residues of the APR, respectively (Fig. 1) (3). DAPPh has no zinc ion, but is activated by cobalt ion (3). In order to identify and characterize the active site of DAPPh, we prepared the mutants of this enzyme and measured their activity. MATERIALS AND METHODS Materials. The DAPPh gene from P. horikoshii (the open reading frame PH0519 amplified using pET11a) was used as a template for constructing mutation products (3). Ala-Ala-Ala(tri-Ala) peptide was purchased from Bachem (Bubendorf, Switzerland). DNA primers were synthesized by Hokkaido System Science (Sapporo, Japan). All the other chemicals and reagents used were of the highest grade commercially available. Construction and expression of the mutant enzymes. The mutation of the DAPPh gene was performed by the PCR (8). The amplified products were digested by NdeI and BamHI and inserted into pET11a cut by the same restriction enzymes. The sequences of the mutated genes were confirmed by an LI-COR Model LIC-4200L(S)-2 sequencer of Aloka (Mitaka, Japan). The recombinant plasmids, which had the mutated DAPPh gene, were expressed in the host E. coli BL21(DE3) codon plus RIL according to the previous method (2). The cultivation was carried out for 4 or 24 h with or without isopropyl-thiogalactopyranoside (IPTG). The concentration of the expressed protein was determined by the Bradford method using a Bio-Rad protein assay kit. Purification of the enzyme. The culture that contained the expressed mutant enzyme was centrifuged at 3000 ⫻ g for 10 min at 4°C. The E. coli pellet was frozen and thawed, and suspended in 10 vol of 50 mM Tris-HCl (pH 7.5)/600 mM NaCl. The suspension was sonicated and centrifuged at 18000 ⫻ g for 10 min at 4°C. The supernatant was heat-treated at 85°C for 15 min, and centrifuged at 18000 ⫻ g for 30 min at 4°C. The supernatant was applied to Phenyl Sepharose of Pharmacia (Uppsala, Sweden), and then to a Hi-Trap Q Sepharose column of Pharmacia. This order was found to be critical, since the application on Phenyl Sepharose with a phosphate buffer

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FIG. 1. Comparison of the amino acid sequences around putative catalytic domain of the deblocking aminopeptidase from P. horikoshii (DAPPh), the deblocking aminopeptidase from Pyrococcus furiosus (DAPPf), and the aminopeptidase from Aeromonas proteolytica (APR). The sequences have been aligned with dashes indicating gaps. Dots (䡠) in DAPPf and APR sequence indicate the same amino acid as that in the DAPPh sequence. The regions conserved among the DAPPh, DAPPf, and APR sequences are indicated by boxes. The asterisks (*) on the sequence indicate the putative active residues.

led to remaining of phosphate ion, which reacted with the cobalt ion. The other conditions were described previously (2). The purity and molecular weight of the mutants were confirmed by SDS-PAGE. The purified enzyme was dialyzed against 50 mM tris-HCl buffer (pH 8.0) and used for the assay. The bound metals of purified enzyme was analyzed with inductively coupled plasma atomic emission spectroscopy (ICP-AES) (model IRIS AP; TJASolutions, Franklin, MA) (3). Assay of the enzyme. The aminopeptidase activity of these enzymes was determined using tri-Ala peptide as a substrate. 0.5 ␮g of the enzyme was incubated at 85°C with the substrate in 100 ␮l of 50 m⌴ N-ethylmorpholine(NEM) buffer (pH 7.5), containing 100 mM NaCl and various concentrations of CoCl 2. The released products were measured by detecting the exposed ␣-amino group using the cadmium-ninhydrin colorimetric method (9). The activity was determined by the optical density at 505 nm using a spectrophotometer. The thermostability of these enzymes was determined by measuring their circular dichroism (CD) by a JASCO (Hachioji, Japan) J-820 spectropolarimeter.

RESULTS AND DISCUSSION The Activity of the Mutants DAP is a novel aminopeptidase which has acylamino acid releasing (deblocking) activity (3). DAPs from P. horikoshii and P. furiosus have two conserved regions with the APR (3) (Fig. 1). Especially some carboxyl residues in these regions are highly conserved among the aminopeptidases (Fig. 1). In the case of APR, it is suggested that Asp117, Glu151 and Glu152 are located at the active site and participate in the activity (6, 7). However, normal aminopeptidase has no deblocking activity. There is no information about the structure and active site of DAP. Therefore, using the information of APR and the conserved region, we prepared three mutant enzymes of DAPPh, in which the carboxyl groups were converted to the corresponding amidated forms (D173N, E205Q and E206Q). The wild type DAPPh was overexpressed by cultivating for 4 h in the presence of IPTG (3). On the other hand, the mutants were overexpressed for 24 h without IPTG. Purification of the mutant enzymes was performed by the same method as that for the wild type. The molecular weight of the mutants was confirmed by SDS-PAGE to be 36.9 kDa, the same as that of the wild type. Analysis by ICP-AES showed that DAPPh contained 1.32 mole of calcium ion per mole of monomer

enzyme protein. The analysis also showed that DAPPh contained less than 0.001 mole of cobalt ion per mole of monomer enzyme protein. This result indicated that no cobalt ion bound to DAPPh. The CD spectra between 200 and 250 nm of the mutants was unchanged between 25 and 95°C (data not shown). Therefore, the thermostability and folding of the enzyme were not changed by the mutations. The K m and k cat values of the mutants were calculated from the measured activity for tri-Ala peptides (Table 1). The K m values of the DAPPhs were not significantly different, indicating that the mutation did not affect the affinity for the substrate. On the other hand, the values of k cat were significantly reduced in the mutants (Table 1). These results indicate that the three carboxyl residues Asp173, Glu205 and Glu206 all contribute to the activity of this enzyme. The X-ray structural analysis of the APR has implied that at least two of the three carboxyl amino acid residues (Asp117, Glu151 and Glu152) are involved in the catalysis by interacting with zinc ions (6, 7). We have demonstrated directly that all three residues are important for the catalytic activity of DAPPh. It can also be supposed that the catalytic domain of DAPPh has structural similarity to APR in spite of the low similarity between the whole sequences.

TABLE 1

K m and k cat of the Wild Type and Mutant DAPPhs Cobalt ion ⫺

⫹

Enzyme

k cat (s ⫺1)

K m (mM)

k cat (s ⫺1)

Wild type D173N E205Q E206Q

2.08 ⫾ 0.43 ⬍0.001 ⬍0.001 ⬍0.001

4.77 ⫾ 0.80 3.40 ⫾ 5.04 8.66 ⫾ 6.42 7.38 ⫾ 5.73

9.01 ⫾ 0.73 0.091 ⫾ 0.064 0.093 ⫾ 0.016 0.226 ⫾ 0.111

Note. The hydrolytic reaction with tri-Ala was measured at 85°C in 50 mM NEM acetate buffer (pH 7.5) containing 100 mM NaCl and with (⫹) or without (⫺) CoCl 2. The CoCl 2(⫹) concentration for the wild type was 2 mM and for the three mutants 8 mM.

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FIG. 2. The effects of cobalt ion for hydrolytic activity of wild type (A) and mutants (C). B and D show Eadie plots of the data in A and C, respectively. Symbols (F, E, ƒ, and Œ) indicate wild type, D173N, E205Q, and E206Q, respectively. U (unit) is defined as releasing of mmol alanine per 1 h by 1 ␮mol enzyme. The hydrolytic reaction was measured at 85°C in 50 mM NEM acetate buffer (pH 7.5), 100 mM NaCl and 2 mM tri-Ala in the presence of various amounts of CoCl 2. ‚ Activity shows the difference in activity between the presence and absence of cobalt ion. [Co] is concentration of cobalt ion (mM).

Effect of Cobalt Ion on the Activity APR activity seems to be related with zinc ions in a co-catalytic unit that contains carboxyl residues (6, 7). We could, however, not find any zinc binding motif in the sequence of DAPPh and Ando et al. (3) showed that DAPPh has no zinc ion but is activated by a cobalt ion. These results suggest that this enzyme has a cobalt binding site that is important for the hydrolytic activity. A change in activity depending on the cobalt ion concentration was examined for the wild type and mutant DAPPhs (Fig. 2). The wild type was strongly activated by cobalt ion. On the other hand, the activity of D173N, E205Q, and E206Q was only slightly increased by cobalt ion (Table 1). We could not detect activity without the cobalt ion for the mutants, whereas the wild type enzyme showed weak activity even without the cobalt ion. In addition, the apparent dissociation constants (K d) of cobalt ion for the wild type DAPPh and its mutants were determined (Fig. 2, Table 2). The K d values of cobalt ion for the three mutants were higher than that

for the wild type by two to three orders of magnitude. These results show that Asp173, Glu205 and Glu206 residues contribute to the affinity for the cobalt ion and activation. Especially, Glu205 and Glu206 strongly participate in the binding of cobalt ion. It is also noted that the shape of the curves in Fig. 2C for the mutants E205Q and E206Q is sigmoidal, whereas for the wild type and D173N it is not. This result suggests that at least two cobalt ions contribute TABLE 2

The Dissociation Constant of Cobalt Ion for DAPPhs Enzyme

K d (mM)

Wild type D173N E205Q E206Q

0.0214 ⫾ 0.0077 1.81 ⫾ 0.404 15.1 ⫾ 11.2 12.3 ⫾ 1.26

Note. The hydrolytic reaction was measured at 85°C in 50 mM NEM acetate buffer (pH 7.5), 100 mM NaCl and 2 mM tri-Ala in the presence of various amounts of CoCl 2.

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to the activity in a co-operative manner. It is interesting that Chevrier et al. proposed a co-catalytic mechanism of the APR activity, in which two zinc ions are involved (7). These zinc ions play equivalent roles during catalysis (6, 7). Alternatively, the results in Fig. 2 and Table 2 suggest that Glu205 and Glu206 of the DAPPh participate in the cobalt ion binding in a similar way, and that the binding contribute to the activity co-operatively. Roderick and Matthews reported on methionine aminopeptidase (MAP) that contained two cobalt ions in its active sites, coordinated with glutamic and aspartic acids (10). Though the DAPPh has no sequence similarity with MAP, activation by two cobalt ions may imply a common mechanism. It is added that we have already succeeded in crystallizing the wild type DAPPh, and the X-ray structure analysis is in progress. It will reveal the similarity and differences of the enzymatic activity of DAP with other metallopeptidases that require more than one metal ion for activity.

3.

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6.

7.

8.

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