Full Paper

Effect of Polycarboxylate Blocks on theAmidase Activity of Trypsin throughComplexation with PEG/PolycarboxylateBlock Ionomers

Atsushi Harada,* Yuriko Yoshioka, Akifumi Kawamura, Chie Kojima,Kenji Kono

The amidase reaction of trypsin, which is a member of the serine proteinase family, isaccelerated by its complexation with block ionomers containing a polycarboxylate block,such as PEG-PAA, PEG-PGA, or PEG-PMA. PEG-PAA and PEG-PGA had similar effects, causing anincrease in the kcat value and a shift in the pHprofile to a lower pH region. On the other hand,PEG-PMA showed not only an increase in thekcat value, but also a decrease in the activationenergy; however, there was no shift in the pHdependence of the initial reaction rate. Suchdifferences might be induced by the differencein pKa values of the polycarboxylate block inblock ionomers.

Introduction

The exocrine pancreas synthesizes, stores, and secretes the

hydrolytic enzymes required for the intestinal digestion

of food. Among these enzymes is the family of serine

proteinases including trypsin, chymotrypsin, elastase, and

kallikrein. Themembers of this family are probably related

by evolution and come from a common ancestral

proteinase; they have retained a similar structure, size,

and function. It is well known that the Asp, His, and Ser

triad, representing common catalytic sites, has an

important role in the enzymatic function of this family.[1]

A. Harada, Y. Yoshioka, A. Kawamura, C. Kojima, K. KonoDepartment of Applied Chemistry, Graduate School of Engin-eering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku,Sakai, Osaka 599-8531, JapanFax: þ81 72 254 9328E-mail: harada@chem.osakafu-u.ac.jp

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In the field of polymer science, complexes of bioactive

compounds, such as enzymes and DNA, with synthetic

polymers, have beenwidely investigated. In particular, the

water-soluble complexes of bioactive compounds with

block ionomers have recently received considerable

attention.[2] As these complexes form a micelle-like

structure, they have utilities as vehicles for bioactive

compounds in the drug delivery field. We have investi-

gated complexes of charged enzymes with block iono-

mers.[3] In such studies, we recently found that the

amidase activity of bovine pancreas trypsin, which is a

member of the serine proteinase family, is facilitated

through the formation of water-soluble complexes with

poly(ethylene glycol)-block-poly(a,b-aspartic acid) (PEG-

PAA).[4] The degree of facilitation was dependent on the

length of the PAA segment as well as the mixing ratio. By

increasing the length of the PAA segment in PEG-PAA, the

amidase reaction of trypsin was facilitated by an even

lower amount of PEG-PAA and the degree of facilitation

DOI: 10.1002/mabi.200600199 339

A. Harada, Y. Yoshioka, A. Kawamura, C. Kojima, K. Kono

Figure 1. Change in the initial reaction rate of complexed trypsinwith mixing ratio. PEG-PAA, *; PEG-PGA, ~; PEG-PMA, &. Sub-strate concentration, 1.4� 10�3 M; trypsin concentration, 4.2�10�6 M; temperature, 25 8C.

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became large. Also, when the amidase reaction of trypsin

was most facilitated by complexing with PEG-PAA, the

complexes of trypsin and PEG-PAA did not form a strict

core-shell architecture, but, rather, formed water-soluble

complexes. The facilitation effect was due to the stabiliza-

tion of the catalytic triad, the Asp-His-Ser residues, by the

carboxylate group of PEG-PAA.

In this study, we evaluated the effect of the type of

polycarboxylate block on facilitation of the amidase

reaction of trypsin. Three kinds of block ionomers, inclu-

ding PEG-PAA, PEG-block-poly(glutamic acid) (PEG-PGA)

and PEG-block-poly(methacrylic acid) (PEG-PMA), all of

which had a comparable polymerization degree of PEG and

the polycarboxylate blocks, facilitated the amidase reac-

tion of trypsin at similarmixing ratio with block ionomers.

Based on an evaluation of the pH and temperature

dependence of the initial reaction rate, it was confirmed

that the pKa value of the polycarboxylate block had a

strong influence on the degree of facilitation of the

amidase activity of trypsin through the stabilization of the

catalytic reaction intermediate.

Experimental Part

Materials

Three types of block ionomer, PEG-PAA [PEG Mn 12 000; PAA Mn

9 300 (DP¼ 68)], PEG-PGA [PEG Mw 12000; PGA Mn 10 600

(DP¼ 70)] and PEG-PMA [PEG Mn 12 500; PMA Mn 6 900

(DP¼ 64)], were used in this study. PEG-PAA and PEG-PGA were

synthesized as previously described.[3a,5] PEG-PMAwas purchased

from Polymer Source, Inc. (Montreal, Canada), and used without

further purification. Bovine pancreas trypsin and L-lysine

p-nitroanilide were purchased from Sigma (St. Louis, MO, USA),

and used without further purification.

Preparation of Trypsin/Block Ionomer Complexes

Trypsin (8 mg �mL�1) and varying concentrations of the block

ionomers in sodium phosphate buffer (10�10�3 M, pH¼ 7.4) were

prepared separately as stock solutions, and then stored under cool

conditions (4 8C) to prevent the autolysis of trypsin. Equal volumes

of trypsin solution and each block ionomer solutionweremixed in

order to have a fixed trypsin concentration (4 mg �mL�1) and

varying block ionomer concentration, and the mixtures were

stored at 4 8C. The mixing ratio of trypsin and block ionomers was

defined as the molar ratio of the number of carboxylate groups in

the block ionomer to the total number of Arg and Lys residues in

trypsin. After 30 min of mixing at 4 8C, the mixtures were used in

experiments. For evaluation of pH dependence, the initial solvent

was changed from sodium phosphate buffer to phosphate

solution, and the pH was checked before and after activity

measurement.

Evaluation of Amidase Activity

The amidase activity of trypsin was evaluated using L-lysine

p-nitroanilide as a substrate, at 25 8C.[6] 25 mL of each complex

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solution, containing 4 mg �mL�1 trypsin, was added to 975 mL of

substrate solution containing 1.4�10�3 M L-lysine p-nitroanilide.

The reaction rates of native and complexed trypsin with block

ionomers were determined by monitoring the change in

absorbance at 410 nm, the wavelength at which the extinction

coefficient of p-nitroaniline is 8 800 cm�1 �M�1, after mixing the

complex and substrate solutions. The initial reaction rate was

determined from the slope of the change in absorbance at 410 nm

between 150 and 250 s after mixing of sample and substrate

solutions.

Results and Discussion

Figure 1 shows the change in the initial reaction rate oftrypsin complexedwith block ionomers. For all of the blockionomers, the initial reaction rate increased with anincrease inmixing ratio. At amixing ratio of 5, the increasein the initial reaction rate ended, and the difference inreaction rate due to the effect of the mixing ratio was notobserved among the block ionomers. This suggests that thenumber of carboxylate groups is an important factor in thefacilitation of the amidase reaction of trypsin and thatthere is no difference in numerical efficiency among blockionomers. Interestingly, the initial reaction rates at amixing ratio of 5 were different: although trypsincomplexed with PEG-PAA showed an initial reaction rateof 19.1� 10�6 M �min�1, trypsin complexed with PEG-PGAor PEG-PMA showed much lower values (12.6 and12.6� 10�6 M �min�1, respectively). That is, complexationwith PEG-PAA apparently facilitates the amidase reactionof trypsin more effectively than PEG-PGA and PEG-PMA.

DOI: 10.1002/mabi.200600199

Effect of Polycarboxylate Blocks on the Amidase Activity of Trypsin through Complexation with PEG/Polycarboxylate . . .

Scheme 1. Acylation reaction mechanism in the catalytic triad of trypsin.

In order to evaluate the differencein the facilitation effect among blockionomers, the enzymatic reaction con-stants of native and complexed trypsin,which were prepared at a mixing ratioof 5, were determined by preparingLineweaver-Burk plots. Both native andcomplexed trypsin had Lineweaver-Burk plots with good linearity (correla-tion coefficients> 0.95), indicating thatthe enzymatic reaction of trypsin canbe kinetically analyzed based on theMichaelis-Menten equation as follows:

Eþ S !k1

k�1

ES!kcat Eþ P

where E is enzyme, S is substrate, P is productand ES is the complex of enzyme and substrate. Theenzymatic reaction constants, the Michaelis constant[Km¼ (k�1þ kcat)/k1] and the catalytic rate constant (kcat)were determined, and kcat/Km was calculated. Theobtained values are summarized in Table 1. The Km valuesfor complexed trypsin were slightly higher than those fornative trypsin indicating that the affinity between trypsinand substrate is apparently decreased by complexationwith block ionomers. This might be due to steric hindranceby block ionomers, because polycarboxylate blocks ofblock ionomers were located around the trypsin moleculeswithin complexes. As typicalmicelleswere not detected bydynamic light scattering measurements in the mixtures oftrypsin and block ionomers at the mixing ratio of 5 andsignificant difference in the complex size was notobserved, a slight difference in Km value might be dueto differences in the catalytic site rather than differencesin complex size. On the other hand, the kcat values weredrastically increased by complexation with the blockionomers. The kcat values for trypsin complexed withPEG-PAA, PEG-PGA, and PEG-PMA were 18, 16, and 30times higher, respectively, than that for native trypsin. In aprevious study, it was reported that this increase in kcatvalue for trypsin complexed with PEG-PAA was attribu-table to the stabilization of the imidazolium ion of the His

Table 1. Enzymatic reaction constants of native and complexed tryp

kcat

minS1

Native trypsin 0.57 (1.00)b)

Trypsin/PEG-PAAa) 9.97 (17.5)

Trypsin/PEG-PGAa) 9.07 (15.9)

Trypsin/PEG-PMAa) 16.8 (29.5)

a)The complexes of trypsin with block ionomers were prepared usin

against native trypsin.

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residue in the catalytic triad of trypsin.[4] The acylationprocess of the amidase reaction of trypsin is shown inScheme 1. At the catalytic site of trypsin, the nucleophi-licity of the hydroxyl group of Ser195 increases through aproton transfer process in the Asp-His dyad, in which thecarboxylate group of Asp102 stabilizes the imidazoliumion of His57. Not only Asp102 of trypsin, but also some ofthe carboxylate groups of PEG-PAA, stabilized the imida-zolium ion of His57 during this reaction process. From theincrease in the kcat values summarized in Table 1, it wasindicated that PEG-PMA might stabilize the imidazoliumion of His57 of trypsin more effectively than PEG-PAA andPEG-PGA.

In order to clarify the effect of different kinds ofpolycarboxylate block on the facilitation of the acylationreaction, the pH dependence and temperature dependenceof the initial reaction rate were evaluated. Figure 2 showsthe pH dependence of the initial reaction rate for nativeand complexed trypsins. The enzymatic activity of trypsinstrongly depends on the pH, because His is a weak cationicresidue and Asp is a weak anionic residue; both exist inequilibrium between protonated and non-protonatedstates. For both the native and complexed trypsins, theinitial reaction rate decreased with decreasing pH. Asexpected, the pH profile of trypsin complexed withPEG-PAA or PEG-PGA was shifted to a lower pH rangecompared with that of the native trypsin. On the other

sin determined from Lineweaver-Burk plots.

Km kcat /Km

10S3M 102 M

S1 �minS1

1.23 (1.00) 4.62 (1.00)

1.60 (1.30) 62.3 (13.5)

2.63 (2.14) 34.5 (7.47)

5.02 (4.08) 33.4 (7.23)

g a mixing ratio of 5; b)Values in parentheses are relative values

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A. Harada, Y. Yoshioka, A. Kawamura, C. Kojima, K. Kono

Figure 2. pH dependence of the initial reaction rate of nativetrypsin (*) and trypsin complexed with PEG-PAA (*), PEG-PGA(~), and PEG-PMA (&). Substrate concentration, 1.4� 10�3 M;trypsin concentration, 4.2� 10�6 M; temperature, 25 8C.

Figure 3. Arrhenius plots of the initial reaction rate of nativetrypsin (*) and trypsin complexed with PEG-PAA (*), PEG-PGA(~), and PEG-PMA (&). The activation energies presented in thetext are the averages of three experiments� SD.

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hand, there was no shift in the pH profile of trypsincomplexed with PEG-PMA; only an increase in the reactionrate was observed. Such a difference in pH profile amongblock ionomers could be explained by the difference inthe pKa values of the polycarboxylate blocks in the blockionomers. The pKa values were determined to be 4.9, 5.0,and 6.7 for PEG-PAA, PEG-PGA, and PEG-PMA, respectively,by acid-base titration at 25 8C. PEG-PMA had a high pKa

value compared with the other block ionomers, PEG-PAAand PEG-PGA. Because of the low pKa values of PEG-PAAand PEG-PGA, a part of the carboxylate group in eachionomer exists in the non-protonated form at pH <6, avalue atwhich native trypsin shows no amidase activity. Anon-protonated carboxylate group could participate in thestabilization of the imidazolium ion of the His residue. As aresult, trypsin complexed with PEG-PAA and PEG-PGAshowed amidase activity at pH <5. However, PEG-PMA,with a higher pKa value, does not have a non-protonatedcarboxylate group at pH<5; thus, no shift in the pH profilewas observed. The shift in the pH profile, reflecting thedifference in the pKa values of block ionomers, indicates aninteraction between the His residue and the carboxylategroup of block ionomers.

Furthermore, the temperature dependence of the initialreaction rate was evaluated in order to discuss theinfluence of complexation with block ionomers onactivation energy. The Arrhenius plots (Figure 3) showgood linearity in the evaluated temperature range from10 8C to 25 8C, not only for native trypsin but also fortrypsin complexed with PEG-PAA, PEG-PGA, or PEG-PMA.The activation energies were determined from the slopesof Arrhenius plots. The activation energies of native

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trypsin and trypsin complexed with PEG-PAA, PEG-PGA,and PEG-PMA were determined to be 22.7� 6.1, 26.9� 1.3,17.5� 1.1 and 11.7� 2.1 kJ �mol�1, respectively; valuesrepresent the average of three experiments� SD. Con-sidering experimental error, the activation energies oftrypsin complexed with PEG-PAA or PEG-PGA were notdifferent from that of native trypsin. By contrast, theactivation energy of trypsin complexedwith PEG-PMAwassignificantly lower than that of native trypsin. Thedifference in activation energy among block ionomersagreed with the difference in kcat values summarizedin Table 1. PEG-PMA, which increased the kcat valueefficiently, produced a decrease in activity energy. As wellas the difference in the pH dependence of initial reactionrate, the difference in kcat values and activation energiescould be explained by the difference in the pKa values ofthe polycarboxylate block in each block ionomer, based onthe assumption that the differences in pKa might also bereflected in the complexes with trypsin. Although therewas no direct structural evidence of an interactionbetween the His residue of the catalytic triad of trypsinand the carboxylate group in the block ionomers, theeffect of complexation with block ionomers on theamidase activity of trypsin is considered to be based onthe acylation reactionmechanism shown in Scheme 1. Thereaction can be summarized as follows: a carboxylategroup of the block ionomer stabilizes the (b) form, which isan intermediate of the acylation reaction, and shifts theequilibrium between (a) and (b) forms towards the (b)form. That is, two types of (b) form co-exist in the case ofcomplexed trypsin. One is formed from the originalAsp-His dyad, and the other is formed from the carboxylate

DOI: 10.1002/mabi.200600199

Effect of Polycarboxylate Blocks on the Amidase Activity of Trypsin through Complexation with PEG/Polycarboxylate . . .

group of the block ionomer and the His residue. Thenucleophilicity of the hydroxyl group of Ser195 increasesthrough a proton transfer process in the carboxylate/imidazolium pair, which depends on the nature of thecarboxylate group. With an increase in the proto-n-withdrawing nature of the carboxylate group, thehydrogen of the hydroxyl group of Ser195 is attracted tothe carboxylate/imidazolium pair. As a result, an increasein the nucleophilicity of the hydroxyl group of Ser195 isinduced. The difference in the proton-withdrawing natureof the carboxylate group could be reflected in thedifference in pKa value. PEG-PMA had higher pKa valuescompared with the other block ionomers, and PEG-PMAhad strong proton-withdrawing nature compared withPEG-PAA and PEG-PGA. Consequently, trypsin complexedwith PEG-PMA showed a higher kcat value and loweractivation energy than trypsin complexed with eitherPEG-PAA or PEG-PGA.

Conclusion

The amidase reaction of trypsin was facilitated by

complexation with block ionomers containing polycar-

boxylate blocks such as poly(a,b-aspartic acid), poly(glu-

tamic acid), and poly(methacrylic acid). All of the block

ionomers induced an increase in the kcat values through

complexation. This increase in kcat values was induced by

the stabilization of the intermediate in the catalytic

reaction of trypsin. During the catalytic reaction process,

some of the carboxylate groups of the block ionomer

interacted with the imidazolium ion of the His residue and

the number of carboxylate/imidazolium pairs increased.

By this numerical effect, the proton transfer process in the

catalytic reaction effectively progressed. Furthermore, a

block ionomer with a relatively high pKa value, PEG-PMA,

induced a decrease in the activation energy of the catalytic

reaction of trypsin. The Asp-His-Ser triad is a common

catalytic site for the family of serine proteases, and the

Asp-His dyad plays an important role in other kinds of

enzymes such as apurinic endonuclease 1, which is a

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humanDNA repair enzyme that cleaves at sites adjacent to

the abasic sites in DNA.[7] The results presented here

suggest that block ionomers with a polycarboxylate block

might have the potential ability to accelerate the

enzymatic function of not only trypsin, but also various

kinds of enzymes through the stabilization of the proton

transfer process in the Asp-His dyad.

Received: September 11, 2006; Revised: November 29, 2006;Accepted: December 4, 2006; DOI: 10.1002/mabi.200600199

Keywords: block copolymers; catalytic triad; enzymes; polycar-boxylate

[1] [1a] D. H. Blow, J. J. Birktoft, B. S. Hartley, Nature 1969, 221,337; [1b] M. W. Hunkapiller, S. H. Smallcombe, D. R. Whitaker,J. H. Richards, J. Biol. Chem. 1973, 248, 8306; [1c] G. Robillard,R. G. Shulman, J. Mol. Biol. 1974, 86, 519; [1d] S. Scheiner,W. N. Lipscomb, Proc. Nat. Acad. Sci. USA 1976, 73,432; [1e] J. L. Markley, I. B. Ibanez, Biochemistry 1978, 17,4627; [1f] A. A. Kossiakoff, S. A. Spencer, Biochemistry 1981,20, 6462; [1g] C. S. Craik, S. Roczniak, C. Largman, W. J. Rutter,Science 1987, 237, 909.

[2] [2a] A. V. Kabanov, V. A. Kabanov,Adv. DrugDelivery Rev. 1998,30, 49; [2b] K. Kataoka, A. Harada, Y. Nagasaki, Adv. DrugDelivery Rev. 2001, 47, 113; [2c] H. Colfen, Macromol. RapidCommun. 2001, 22, 219; [2d] A. Harada, K. Kataoka, Prog.Polym. Sci. 2006, 31, 949.

[3] [3a] A. Harada, K. Kataoka, Macromolecules 1998, 31, 288;[3b] A. Harada, K. Kataoka, Langmuir 1999, 15, 4208;[3c] A. Harada, K. Kataoka, J. Am. Chem. Soc. 1999, 121,9241; [3d] A. Harada, K. Kataoka, J. Controlled Release 2001,72, 85; [3e] A. Harada, K. Kataoka, J. Am. Chem. Soc. 2003, 125,15301.

[4] A. Kawamura, Y. Yoshioka, A. Harada, K. Kono, Biomacromo-lecules 2005, 6, 627.

[5] H. Cabral, N. Nishiyama, S. Okazaki, H. Koyama, K. Kataoka,J. Controlled Release 2005, 101, 223.

[6] B. F. Erlanger, N. Kokowsky, W. Cohen, Arch. Biochem. Biophys.1961, 96, 271.

[7] D. F. Lowry, D. W. Hoyt, F. A. Khazi, J. Bagu, A. G. Lindsey,D. M. Wilson, III, J. Mol. Biol. 2003, 329, 311.

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