Enzyme and Microbial Technology 75–76 (2015) 10–17

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Mechanism of papain-catalyzed synthesis of oligo-tyrosine peptides Jun Mitsuhashi, Tsutomu Nakayama, Asako Narai-Kanayama ∗ Graduate School of Veterinary Medicine and Life Science, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 180-8602, Japan

a r t i c l e

i n f o

Article history: Received 30 January 2015 Received in revised form 30 March 2015 Accepted 31 March 2015 Available online 28 April 2015 Keywords: Peptide synthesis Oligo-tyrosine Papain

a b s t r a c t Di-, tri-, and tetra-tyrosine peptides with angiotensin I-converting enzyme inhibitory activity were synthesized by papain-catalyzed polymerization of l-tyrosine ethyl ester in aqueous media at 30 ◦ C. Varying the reaction pH from 6.0 to 7.5 and the initial concentration of the ester substrate from 25 to 100 mM, the highest yield of oligo-tyrosine peptides (79% on a substrate basis) was produced at pH 6.5 and 75 mM, respectively. In the reaction initiated with 100 mM of the substrate, approx. 50% yield of insoluble, highly polymerized peptides accumulated. At less than 15 mM, the reaction proceeded poorly; however, from 30 mM to 120 mM a dose-dependent increase in the consumption rate of the substrate was observed with a sigmoidal curve. Meanwhile, each of the tri- and tetra-tyrosine peptides, even at approx. 5 mM, was consumed effectively by papain but was not elongated to insoluble polymers. For deacylation of the acyl-papain intermediate through which a new peptide bond is made, l-tyrosine ethyl ester, even at 5 mM, showed higher nucleophilic activity than di- and tri-tyrosine. These results indicate that the mechanism through which papain polymerizes l-tyrosine ethyl ester is as follows: the first interaction between papain and the ester substrate is a rate-limiting step; oligo-tyrosine peptides produced early in the reaction period are preferentially used as acyl donors, while the initial ester substrate strongly contributes as a nucleophile to the elongation of the peptide product; and the balance between hydrolytic fragmentation and further elongation of oligo-tyrosine peptides is dependent on the surrounding concentration of the ester substrate. © 2015 Elsevier Inc. All rights reserved.

1. Introduction l-Tyrosine (Tyr) is classified as a non-essential amino acid because it is formed in vivo by hydroxylation of dietary phenylalanine (Phe), except for in the case of some newborns. Thus, Tyr is an essential amino acid in patients with phenylketonuria, a genetic disorder in which Phe cannot be normally metabolized. Since the addition of Tyr can sustain synthesis of catecholamines, the potential usefulness of supplementary Tyr for the treatment of phenylketonuria, Parkinson’s disease, and acute stress has been investigated [1,2]. However, the use of free Tyr is limited due to its low solubility and questionable efficacy. In contrast to free Tyr, dipeptides containing Tyr are generally water-soluble, therefore the development of facile operations for the synthesis of

Abbreviations: ACE, angiotensin I-converting enzyme; BA, N␣-benzoyl-larginine; BAEE, N␣-benzoyl-l-arginine ethyl ester; BA-Tyr, N␣-benzoyl-l-arginyl-ltyrosine; DP, degree of polymerization; RP-HPLC, reverse-phase high-performance liquid chromatography; Tyr-OEt, l-tyrosine ethyl ester; Tyr-OH, free l-tyrosine. ∗ Corresponding author. Tel.: +81 422 31 4151; fax: +81 422 51 9984. E-mail address: [email protected] (A. Narai-Kanayama). http://dx.doi.org/10.1016/j.enzmictec.2015.03.007 0141-0229/© 2015 Elsevier Inc. All rights reserved.

Tyr-containing peptides is desired [2–4]. Furthermore, a number of Tyr-containing di- or tri-peptides from several food protein sources exhibit angiotensin I-converting enzyme (ACE, EC. 3.4.15.1) inhibitory activity, which contributes to the regulation of blood pressure [5–8]. Among them, Tyr-Tyr is isolated from royal jelly treated with protease [8]. According to some research [9,10], TyrTyr is effectively transported through PEPT1, a proton-coupled peptide transporter expressed on the intestinal epithelium. Thus, it is worth evaluating the bioactivity and safety of Tyr-Tyr for the development of novel therapeutic and/or nutraceutical products. Pure dipeptides synthesized by chemical methods are commercially available; however, they are generally too expensive to be readily used. In the chemical synthesis of Tyr-containing peptides, not only the N- and C-terminal groups in amino acids but also other functional groups in the side chains are commonly protected [11–14]. Subsequently, coupling reaction and acid-catalyzed removal of the protecting groups lead to unwanted side reactions and the formation of byproducts. To overcome the problems of chemical peptide synthesis, protease-catalyzed reactions have been attracting considerable attention [14–18]. In protease-catalyzed peptide synthesis, the high specificity and high reactivity under mild conditions, which are characteristics of

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enzymes, can reduce the number of operational steps required for protection–deprotection and avoid side reactions such as racemization. The acyl group of a substrate specific to the S subsite region in a protease is transferred to an added nucleophile with a high affinity for the S’ subsite in the catalyst through the acyl-enzyme intermediate [18–22]. The deacylation by amino components such as amino acid derivatives or peptides is called aminolysis, generating a new peptide bond. In an aqueous reaction, hydrolysis is unavoidable due to the presence of water. Temperature, pH and nucleophile concentration are important parameters that affect peptide yield by regulating the rate of aminolysis by reactive nucleophiles with deprotonated amino group. With respect to the efficient synthesis of Tyr-Tyr, NaraiKanayama et al. [23] previously proposed the two-step enzymatic reaction, in which papain (EC 3.4.22.2)-catalyzed synthesis of Tyrpolymers from l-tyrosine ethyl ester (Tyr-OEt) in a buffer solution [24] was followed by their hydrolytic cleavage by ␣-chymotrypsin (EC 3.4.21.1) in aqueous DMSO media [23]. Overall, these two reactions were carried out at 25 ◦ C and pH 7.5. Starting with 100 mM of Tyr-OEt, the final reaction products contained di- and tri-Tyr peptides in good yield, reaching 65% on an initial ester substrate basis [23]. Each of these two oligo-Tyr peptides exhibited a mixture of competitive and noncompetitive inhibitions of ACE from rabbit lung, with IC50 values of 34 ␮M and 48 ␮M, respectively [21]. However, the two-step enzymatic reaction proceeds slowly, at least 4 days. Furthermore, centrifugation is necessary between the two reactions to collect the water-insoluble Tyr-polymers with degrees of polymerization (DPs) of between 5 and 10, and DMSO is used as a solvent to dissolve Tyr-polymers. The 2nd reaction step needs a substantial amount of ␣-chymotrypsin to bring its hydrolytic activity enough in a 50% DMSO/buffer solution. Ultimately, DMSO and some peptide fragments derived from autoprocessing of ␣chymotrypsin must be removed from the oligo-Tyr peptides. Therefore, the development of a high throughput method for the synthesis of oligo-Tyr peptides using only papain-catalyzed polymerization of Tyr-OEt without both DMSO and ␣-chymotrypsin has been anticipated. In the present study, we investigated the effects of reaction temperature, pH and initial Tyr-OEt concentration on the yields of oligo-Tyr peptides. Also, we kinetically analyzed the mechanism through which papain polymerized Tyr-OEt and elongated oligo-Tyr peptides to Tyr-polymer.

initiated by mixing with an activated enzyme solution at a 10:1 volume ratio, with a final papain concentration of 30 ␮M, and the reaction proceeded mildly enough to allow us to temporally analyze the reaction products. Aliquots of the reaction mixture were withdrawn at appropriate intervals, added into 1/5 volume of 5 M HCl, and then mixed well to make l-tyrosine (Tyr-OH), a hydrolysis product of Tyr-OEt, soluble and distinctive from insoluble Tyr-polymers. These sample solutions were kept on ice to prevent the ester group hydrolysis of Tyr-OEt and products. These were used for the quantitative analysis of the reaction products by RPHPLC, which was carried out using a Hitachi HPLC system (L-7100 pump, L-7400 UV detector, and L-7200 autosampler) equipped with a SUS line filter (GL Science, Tokyo, Japan) and a Cadenza CD-C18 (4.6 mm × 50 mm) column (Imtakt, Kyoto, Japan). Tyr-OEt, Tyr-OH, and oligo-Tyr peptides with DP 2, 3 and 4 were eluted using a linear gradient (0–50% for 40 min) of methanol/0.1% TFA at a flow rate of 0.5 mL/min, and detected at 280 nm. The concentration of each component on the basis of Tyr residues was determined from peak areas as described previously [20], except for the analysis of chromatograms, which used the data processing software Chromato-PRO (Run Time Corporation, Kanagawa, Japan).

2. Materials and methods

2.4. Kinetic analysis of papain-catalyzed acyl transfer reaction

2.1. Materials l-Tyrosine ethyl ester (Tyr-OEt) hydrochloride was purchased from Tokyo Chemical Industry (Tokyo, Japan). Papain from papaya latex, a 2× crystallized suspension in sodium acetate, pH 4.5, and N␣-benzoyl-l-arginine ethyl ester (BAEE) were purchased from Sigma–Aldrich (St Louis, USA). The molecular concentration of papain was determined from its absorbance at 278 nm using the value of E1%, 1 cm, 278 nm = 25.0 and the molecular weight of 23,700 [25]. Chemically synthesized Tyr-Tyr, >95% purity, was purchased from Thermo Fisher Scientific (Ulm, Germany). Trifluoroacetic acid (TFA) was purchased from Kanto Chemical (Tokyo, Japan). All other reagents used were of analytical grade.

2.2. Papain-catalyzed polymerization of Tyr-OEt Unless otherwise specified, papain-catalyzed polymerization of Tyr-OEt was performed using 0.2 M Na-phosphate buffer, pH 6.5, containing 1 mM DTT at 30 ◦ C without pH control. It was confirmed that the reaction pH changed by only 0.2–0.4 during the reaction. Papain solution was prepared at 330 ␮M in 11 mM DTT and was then activated by incubation at 30 ◦ C for 10 min before the reaction. Tyr-OEt was dissolved in 0.22 M Na-phosphate buffer and the pH of this substrate solution was adjusted to the predetermined value with a NaOH solution. After preincubation of the substrate solution at a given temperature for 1 min, the reaction was

2.3. Preparation of oligo-Tyr peptides with DP 2, 3, and 4 The acidified reaction mixture (as described in Section 2.2) was centrifuged at 10,000 × g for 30 min at 25 ◦ C, and the supernatant was mixed with 10 M NaOH at a 17:3 volume ratio. This alkaline de-esterification of residual Tyr-OEt for 30 min at 25 ◦ C in the dark was stopped by mixing with 1/5 volume of 5 M HCl. After the removal of sediment by centrifugation, the solution was loaded onto a NOBIAS RP-OD1 cartridge (Hitachi High Technologies, Tokyo, Japan). The absorbed matters were eluted using 50% ethanol and then evaporated. From this concentrate, which mainly contained Tyr-Tyr (DP2), Tyr-Tyr-Tyr (DP3) and tetra Tyr peptide (DP4), each of the oligo-Tyr peptides was purified by RP-HPLC using a preparative column (InertSustainTM C18 (7.6 mm × 150 mm); GL Science) with a linear gradient (4.5–54% for 35 min) of acetonitrile/0.1% TFA at a flow rate of 0.5 mL/min. The retention time of DP 2 was confirmed using the commercially obtained Tyr-Tyr.

Papain-catalyzed acyl transfer reactions were conducted using 1 ␮M papain and 100 mM of BAEE as an acyl donor in 0.2 M Naphosphate buffer, pH 6.5, containing 1 mM DTT at 30 ◦ C. BAEE of 110 mM and a specified concentration of nucleophile such as TyrOEt or oligo-Tyr peptides were dissolved in 0.22 M Na-phosphate buffer and the pH was adjusted to 6.5 with a NaOH solution. After preincubation of the substrate solution at 30 ◦ C for 1 min, the acyl transfer reaction was initiated by mixing with an activated papain solution of 11 ␮M at a 10:1 volume ratio. Aliquots (each 100 ␮L) of the reaction mixture were withdrawn at intervals of 5 min within 25 min, added to 1.5 mL of 0.05 M HCl and kept on ice. RP-HPLC analysis of BAEE, N˛-benzoyl-l-arginine (BA), Tyr-OEt, DP2 and synthetic peptides (BA-Tyr-OEt or BA-DP2) were performed using a Cadenza CD-C18 (4.6 mm × 50 mm) column (Imtakt) with linear elution (0–54–90–90% of methanol/0.1% TFA: 0–30–40–45 min) at a flow rate of 0.5 mL/min, and all eluted components were detected at 260 nm (Fig. 1A and B). DP3 and BA-DP3 were analyzed using an InertSustainTM C18 (4.6 mm × 75 mm) column (GL Science) under a different elution condition (0–0–54–90–90% of methanol/0.1% TFA: 0–5–30–40–45 min) (Fig. 1 C). Products other than BA and BA-X peptides (X: Tyr-OEt, DP2 or DP3) were not detected under the conditions used, as shown in Fig. 1. Velocities

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3. Results and discussion 3.1. Effects of reaction temperature, pH, and initial Tyr-OEt concentration on yield of oligo-Tyr peptides In protease-catalyzed peptide synthesis, there are various factors responsible for the balance between hydrolysis and aminolysis that affect the reaction efficiency and peptide yield. For example, an increase in temperature not only accelerates the enzymatic reaction but also activates water molecules, enhancing hydrolysis [26]. In the absence of papain, an increase in temperature induced auto-hydrolysis of Tyr-OEt (Table 1, papain (−)). Conversely, in the presence of papain, the amount of released Tyr-OH decreased, except for at 50 ◦ C (Table 1, papain (+)). Elevated reaction temperature up to 40 ◦ C increased DP2 and DP3 with an accompanying decrease in DP4 and residual Tyr-OEt; however, the yield of insoluble Tyr-polymer with DP≥5 did not significantly decrease. Although the yield of oligo-Tyr peptides exceeded 41% at 50 ◦ C, a heatdependent byproduct was produced, which was eluted near DP2 in the RP-HPLC analysis (data not shown). These results suggested that at higher temperatures papain rapidly polymerized Tyr-OEt before non-enzymatic hydrolysis of the ester substrate and that increasing temperature could not sufficiently suppress the accumulation of insoluble Tyr polymers. In the present study, the following reactions were conducted at 30 ◦ C to minimize auto-hydrolysis of Tyr-OEt and some side reactions as well as heat inactivation of papain. Low pH appears preferable because substrates with protonated amino group cannot participate in peptide synthesis as a nucleophile [27]. Indeed, lowering the reaction pH from 7.5 to 6.5 significantly increased DP2, while DP3, DP4 and Tyr-polymers slightly decreased (Table 2). However, in the reaction at pH 6.0 about 25% of Tyr-OEt remained even after a 72-h reaction (Table 2), suggesting that pH below 6.5 was outside of the optimum pH range of the reaction. In addition to the effects of decreased pH on peptide yields, the effects of initial Tyr-OEt concentration were also investigated at pH 6.5. Decrease in the initial concentration of the substrate increased oligo-Tyr peptides as previously observed at pH 7.5 [23]; however, the change in product yields was much more drastic at pH 6.5, where little Tyr-polymer accumulated in the reaction mixture at an initial Tyr-OEt concentration of 75 mM (1.8%), giving the best yield (79%) of oligo-Tyr peptides (Table 3). The difference in product yields between reactions started with 75 mM and 100 mM of Tyr-OEt was surprisingly large. In order to understand the reasons why further elongation of oligo-Tyr peptides proceeded greatly at high concentrations of the ester substrate, we investigated the mechanisms through which papain catalyzed the polymerization of Tyr-OEt. 3.2. Temporal analysis of papain-catalyzed reaction with Tyr-OEt Fig. 1. Chromatograms in RP-HPLC analysis of nucleophilic activity of Tyr-OEt (A), DP2 (B), and DP3 (C). “Product” means BA-X peptides synthesized by papain with BAEE and each of the nucleophiles.

of BAEE hydrolysis (Vh ) and aminolysis (Va ) were calculated from rates of increase in the peak area of BA and decrease in that of nucleophile, respectively. However, BA and DP2 could not be separately analyzed because they showed approximately the same retention times, even with a longer column. Taking into account that BA-DP2 was minimally detected (Fig. 1B), we propose that the temporal increase in peak area including both BA and DP2 reflected BAEE hydrolysis.

Temporal reduction of Tyr-OEt and production of oligo-Tyr peptides were compared between reactions started with 50 mM and 100 mM of the ester substrate. Within 1-h or 1.5-h reaction, TyrOEt was consumed with a small release of Tyr-OH (Fig. 2A and B), suggesting that most of the ester substrate was used for peptide synthesis rather than hydrolysis. During the early reaction period, it was observed that insoluble products accumulated only in the reaction mixture initiated with 100 mM Tyr-OEt. Amounts of DP3 and DP4 in the reaction of 100 mM Tyr-OEt increased prior to DP2 production (data expressed as open circles in Fig. 2C, D and E), and they were almost twice as large as those in the 50 mM reaction (data expressed as closed circles in Fig. 2C, D and E). Papain is an endoprotease possessing an S2 subsite which specifically accepts a hydrophobic, large residue in the substrate [28]. Thus, the DP2,

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Table 1 Distribution of Tyr residue after incubation of 100 mM Tyr-OEt in the presence (+) or absence (−) of 30 ␮M papain in 0.2 M Na-phosphate buffer, pH 7.5, at various temperatures for 72 h. Reaction temperature (◦ C)

Distribution of Tyr residue (mM) Tyr-OH

Tyr-OEt

DP2

DP3

DP4

DP5

Papain (−)

25 30 40 50

70.9 ± 3.8 86.8 ± 2.5 98.2 ± 4.6 99.7 ± 1.7

29.1 ± 1.5 13.2 ± 0.4 1.8 ± 0.1 0.3 ± 0.0

– – – –

– – – –

– – – –

– – — –

Papain (+)

25 30 40 50

19.4 ± 6.3 16.0 ± 0.8 14.6 ± 0.7 15.7 ± 0.9

5.2 ± 4.5 0.6 ± 0.6 0.0 ± 0.0 0.0 ± 0.0

16.1 ± 4.6 20.7 ± 2.9 26.3 ± 1.6 22.4 ± 1.8

6.4 ± 0.6 7.6 ± 0.1 9.2 ± 0.4 16.5 ± 0.9

2.9 ± 1.4 1.7 ± 0.7 0.7 ± 0.0 2.8 ± 0.0

50.1 ± 8.2 53.5 ± 1.2 49.2 ± 2.7 42.6 ± 3.6

Data are indicated as the mean ± SD (n = 3). Table 2 Distribution of Tyr residue after a 72-h reaction of 100 mM Tyr-OEt with 30 ␮M papain in 0.2 M Na-phosphate buffer, at various pHs and 30 ◦ C. pH

6 6.5 7 7.5

Distribution of Tyr residue (mM) Tyr-OH

Tyr-OEt

DP2

DP3

DP4

DP5

15.3 ± 3.2 14.8 ± 0.4 18.5 ± 0.9 16.0 ± 0.8

25.4 ± 3.8 4.0 ± 0.8 0.4 ± 0.3 0.6 ± 0.6

24.8 ± 4.1 31.9 ± 0.9 27.8 ± 0.3 20.7 ± 2.9

6.8 ± 1.1 6.4 ± 0.4 5.2 ± 0.2 7.6 ± 0.1

0.4 ± 0.1 1.5 ± 0.5 0.6 ± 0.2 2.0 ± 0.4

27.4 ± 12.3 41.2 ± 2.1 47.6 ± 1.5 53.5 ± 1.2

Data are indicated as the mean ± SD (n = 3).

DP3, and DP4 we analyzed seemed to be produced by enzymatic hydrolysis of either peptide bond or ester bond of the oligo-Tyr ethyl esters, such as Tyr-Tyr-OEt (DP2-OEt), an initially synthesized peptide, as well as DP3-OEt and DP4-OEt. Because the ester forms of oligo-Tyr peptides could not be detected by RP-HPLC, their affinity towards the S subsite region in papain was probably so high that papain might consume them very rapidly as acyl donors [25]. Therefore, in the early reaction, papain-catalyzed polymerization of Tyr-OEt preferentially occurred, resulting in synthesis of the ester form of oligo-Tyr peptides; however, both their amounts and DPs as well as their elongation-hydrolysis balance seemed to be strongly dependent on the initial substrate concentration. On the other hand, increase in DP3 and DP4 plateaued between 1.5 and 3 h (Fig. 2D and E), and there was a subsequent gradual reduction (Fig. 2D and E). Despite the fact that about 50 mM TyrOEt still remained at 3 h, the Tyr-OEt consumption rate slowed in the 100 mM reaction (Fig. 2A). In addition, between 3 and 6 h, Tyr residues derived from the increased Tyr-OH (3 mM) and DP2 (5 mM) did not compensate for those of the decreased Tyr-OEt (20 mM), DP3 (7 mM) and DP4 (7 mM). With these results, it is proposed that co-existence of abundant Tyr-OEt and oligo-Tyr peptides in the reaction mixture allowed papain-catalyzed peptide elongation. Using purified oligo-Tyr peptides, we confirmed that papain did not react with DP2, in which both N- and C-terminal groups are closely located (data not shown), but effectively reacted with DP3 and DP4 even at 5.8 mM (corresponding to 17.3 mM of Tyr residues, Fig. 3A) and 3.4 mM (13.6 mM of Tyr residues, Fig. 3B), respectively.

The longer the oligo-Tyr peptide length, the faster the papaincatalyzed reaction proceeded. This might be due to the nature of papain as an endoprotease with an S2 subsite highly specific to a hydrophobic large residue in the substrate. However, insoluble Tyr-polymers did not occur under such conditions. Consumption of DP3 accompanied the remarkable increase in DP2 with a slight release of DP4 and Tyr-OH (Fig. 3A), suggesting that the peptide bond between 2nd and 3rd Tyr residues in DP3 was not simply hydrolyzed. At the 10-min reaction mark, only a slight peak of DP5 was detected in RP-HPLC (data not shown). Thus, DP3 probably acted as both an acyl donor and a nucleophile for peptide elongation, resulting in synthesis of DP5. Further enzymatic aminolysis and hydrolysis of DP5 might occur, releasing DP2 and DP3 as follows: Y-Y∼Y-OH + Y-Y-Y-OH → Y-Y-Y-Y-Y-OH + Y-OH

(1)

Y-Y∼Y-Y-Y-OH + Y-Y-Y-OH → Y-Y-Y-Y-Y-OH + Y-Y-Y-OH (2) Y-Y∼Y-Y-Y-OH → Y-Y-OH + Y-Y-Y-OH

(3)

where Y is a Tyr residue and tilde (∼) indicates a scissile bond. Meanwhile, papain-catalyzed consumption of DP4 was faster than that of DP3. DP4 rapidly decreased with the increase of DP2 and DP3 as main products (Fig. 3B). Since the amount of DP3 was larger than that of DP2, while Tyr-OH was slightly released, it was suggested that DP4 became a highly specific acyl donor for papain but was not simply hydrolyzed, contributing to peptide elongation as follows: Y-Y∼Y-Y-OH + Y-Y-Y-Y-OH → Y-Y-Y-Y-Y-Y-OH + Y-Y-OH (4) Papain was likely to react more effectively with DP6 at the peptide bond between 3rd and 4th Tyr residues as a scissile target, because this position is distant from both N- and C-terminal ends and there are seven subsites in the extended active site of papain, including S1 -S4 and S1  –S3  , of which S2  and S3  as well as S2 have preference for hydrophobic large residues [29]. Although neither DP6 nor DP7 were detected by RP-HPLC, further peptide elongation and cleavage would occur as follows: Y-Y-Y∼Y-Y-Y-OH + Y-Y-Y-Y-OH → Y-Y-Y-Y-Y-Y-Y-OH + Y-Y-Y-OH

(5)

Table 3 Distribution of Tyr residue after a 72-h reaction initiated with various concentrations of Tyr-OEt and 30 ␮M papain in 0.2 M Na-phosphate buffer, pH 6.5, at 30 ◦ C. Initial concentrations of Tyr-OEt (mM)

25 50 75 100 Data are indicated as the mean ± SD (n = 3).

Product concentration (mM) Tyr-OH

Tyr-OEt

DP2

DP3

DP4

Ppt

5.2 ± 0.6 7.7 ± 0.6 10.5 ± 0.5 14.8 ± 0.4

0.2 ± 0.1 1.6 ± 2.1 3.6 ± 5.4 4.0 ± 0.8

16.2 ± 0.7 33.6 ± 0.7 50.6 ± 0.9 31.9 ± 0.9

2.0 ± 1.1 4.2 ± 0.4 7.5 ± 0.2 6.4 ± 0.7

0.1 ± 0.1 0.4 ± 0.5 1.1 ± 0.2 1.5 ± 0.5

1.4 ± 0.8 2.6 ± 2.1 1.8 ± 1.5 41.2 ± 2.1

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Fig. 2. Temporal consumption of Tyr-OEt (A) and production of Tyr-OH (B), DP2 (C), DP3 (D) and DP4 (E). Tyr-OEt was reacted with 30 ␮M papain in 0.2 M Na-phosphate buffer, pH 6.5, at 30 ◦ C. Initial Tyr-OEt concentrations were 100 mM ( ) and 50 mM (䊉).

Y-Y-Y∼Y-Y-Y-OH → Y-Y-Y-OH + Y-Y-Y-OH

(6)

Therefore, it was suggested that papain specifically used these oligo-Tyr peptides as acyl donors and produced shorter peptide lengths through a complicated pathway not simply consisting of hydrolysis, under conditions of low surrounding Tyr-OEt concentration. 3.3. Effects of Tyr-OEt concentration on balance between elongation and cleavage of oligo-Tyr peptides by papain Effects of Tyr-OEt concentration on the initial rates of papaincatalyzed consumption of the ester substrate and synthesis of oligo-Tyr peptides were investigated. As a result, the substrate concentration did not correlate with the consumption rate in a Michaelis–Menten manner (Fig. 4A). Papain did not use Tyr-OEt at lower than 15 mM but consumed it dose-dependently from 30 to 60 mM. The correlation curve was sigmoidal from 60 to 120 mM, and the consumption rate was lowered at 150 mM. The rate of

early synthesis of DP3 and DP4 exceeded that of DP2 in the reaction of Tyr-OEt at greater than 60 mM (Fig. 4B), where insoluble Tyr-polymers were observed in the reaction mixture. These results indicated that papain acylation by Tyr-OEt was a rate-limiting process in the papain-catalyzed polymerization of Tyr-OEt. In contrast to this monomer ester, the oligomers once synthesized could be highly effective acyl donors, as described above. Therefore, there was a strong possibility that Tyr-OEt at a higher concentration was used preferentially as a nucleophile and contributed to the rapid elongation of oligomers. The sigmoidal curve shown in Fig. 4A might reflect that papain-catalyzed consumption of Tyr-OEt was again enhanced by the removal of polymerized peptide products as precipitates from the surrounding environment of the enzyme. Although the reason why excessive amounts of Tyr-OEt inhibited reaction progression remains unclear, a large amount of the monomer ester would be wasted in the synthesis of oligo-Tyr peptides. In order to verify the above possibility, the nucleophilic activity of Tyr-OEt was examined using BAEE, a specific acyl donor, for which the Km for the esterase activity of papain was 33.6 mM

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Fig. 4. Effects of initial Tyr-OEt concentration on the rate of substrate consumption (䊉 in A) and synthetic rates of Tyr-OH ( ), DP2 (), DP3 ( ), and DP4 () (B). Reactions were conducted with 30 ␮M papain in 0.2 M Na-phosphate, pH 6.5, at 30 ◦ C for 50 min. Data are indicated as the mean ± SD (n = 3). Fig. 3. Temporal analysis of reactants in the papain-catalyzed reaction with DP3 (A) and DP4 (B). DP3 (5.8 mM) and DP4 (3.4 mM) were reacted with 30 ␮M papain in 0.2 M Na-phosphate buffer, pH 6.5, at 30 ◦ C for 80 min. Temporal changes in concentrations of Tyr-OH () and oligo-Tyr peptides of DP2 (), DP3 ( ), and DP4 () are shown.

at pH 6.5 (data not shown). In the presence of Tyr-OEt, even at 5 mM, hydrolysis of BAEE was suppressed and BA-Tyr-OEt was effectively synthesized by aminolysis (Fig. 5A). The ratio of Va /Vh was dose-dependently elevated (Fig. 5B). Catalytic constant values for aminolysis with Tyr-OEt (Fig. 5A) were much larger than those shown in Fig. 3A, indicating that papain utilized Tyr-OEt effectively as a nucleophile; however, formation of the acyl-enzyme intermediate with the ester substrate was a slow process. However, DP2 at 5 mM neither suppressed hydrolysis of BAEE nor acted as a nucleophile, while DP3 showed weaker nucleophilic activity than Tyr-OEt (Fig. 6). The catalytic constant value for aminolysis with DP3 was about 30 times greater than that estimated in the papain-catalyzed reaction with DP3 alone (Fig. 3A). This supported the speculation described in Equations 1 and 2 that papain uses DP3 not only as an acyl donor but also as a nucleophile. Both formation of the acyl-enzyme intermediate and aminolysis with such

oligo-Tyr peptides might result in transpeptidation (e.g. Eqs. (1), (2), (4) and (5)). In conclusion, the proposed mechanism of papain polymerization of Tyr-OEt is depicted in Fig. 7, where the first interaction between papain and Tyr-OEt is a rate-limiting step, and then, oligoTyr peptides synthesized early in the reaction are specifically used as acyl donors by the catalyst. When the reaction was initiated with a low concentration of Tyr-OEt (Fig. 7A), almost all of the ester substrate was converted into oligo-Tyr peptides and their esters. This condition, owing to the low concentration of early peptide products, prevents further elongation but allows for their papaincatalyzed hydrolytic fragmentation (e.g. Eqs. (3) and (6)). On the other hand, with an initial high concentration of Tyr-OEt (Fig. 7B), the reaction mixture contains both sufficient Tyr-OEt and a certain amount of the early peptide products. This co-existence allows for further enzymatic elongation of peptides by either aminolysis with Tyr-OEt or transpeptidation. Such conditions cannot prevent synthesis of insoluble Tyr-polymers. According to the scheme, a lower concentration of Tyr-OEt is favorable for papain-catalyzed synthesis of oligo-Tyr peptides with good yield. In this study, the highest yield (79%) of oligo-Tyr peptides occurred in the reaction

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Fig. 7. Proposal of mechanisms through which papain polymerizes Tyr-OEt when the initial concentration of Tyr-OEt is low (A) or high (B). Both reduction of the substrate and increase in oligomers occur in parallel during an early period of the reaction. Direction of the reaction, i.e., production of oligomers or insoluble polymers, is strongly dependent on the initial concentrations of Tyr-OEt.

order to use papain effectively for the synthesis of oligo-Tyr peptides, application of the catalyst immobilized on an insoluble carrier into the reaction represents a promising strategy. References

Fig. 5. Effects of Tyr-OEt concentration on the catalytic constants of papaincatalyzed hydrolysis (Vh ) of BAEE ( in A) and aminolysis (Va ) for synthesis of BA-Tyr-OEt (䊉 in A), and on Va /Vh ratio ( in B). BAEE (100 mM) was reacted with 1 ␮M papain and 0–40 mM Tyr-OEt in 0.2 M Na-phosphate buffer, pH 6.5, at 30 ◦ C for 40 min. Data are indicated as the mean ± SD (n = 3).

Fig. 6. Comparison of nucleophilic activities between DP2 and DP3. BAEE (100 mM) as an acyl-donor and 5 mM of Tyr-OEt, DP2 or DP3 as nucleophiles were reacted with 1 ␮M papain in 0.2 M Na-phosphate buffer, pH 6.5, at 30 ◦ C for 40 min. Catalytic constants of BA production (open bar) and peptide synthesis (closed bar) are shown. Data are indicated as the mean ± SD (n = 3).

with 75 mM Tyr-OEt at pH 6.5, which was much more than the 65% generated by our previously reported two-step enzymatic reaction [22]. Understanding the reasons why further elongation of oligo-Tyr peptides vigorously proceeded at high concentrations of Tyr-OEt affords the effective use of both enzyme and substrate. In

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Mechanism of papain-catalyzed synthesis of oligo-tyrosine peptides.

Di-, tri-, and tetra-tyrosine peptides with angiotensin I-converting enzyme inhibitory activity were synthesized by papain-catalyzed polymerization of...
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