Ostrich pepsins I and II: A kinetic and thermodynamic investigation

~

lnl J. Bio~hem. ('el/Biol. Vol. 27, No. 12, pp. 1293 1302. 1995

Pergamon

1357-2725(95)00092-5

Copyright ~ 1995 ElsevierScienceLtd Printed in Great Britain. All rights reserved 1357-2725/95 $9.50 + 0.00

Ostrich Pepsins I and II: A Kinetic and Thermodynamic Investigation BRETT

I. PLETSCHKE, RYNO J. NAUDI~,* WILLEM OELOFSEN

Department 0/ Biochemismv, Uni1"ersitv Of Port Elizabeth, P.O. Box 1600, Port Elizabeth 6000, South A~'ica Two forms of ostrich pepsin have been purified, ostrich pepsins 1 and II. The aim of this study was to characterize these pepsins in terms of temperature and alkaline stability, temperature and pH optima, and to investigate their kinetic properties. The effect of pH and temperature on ostrich pepsins was studied using the haemoglobin assay method. The hydrolysis of a synthetic hexapeptide substrate Leu-Ser-Phe-(NO2)-Nle-Ala-Leu-OMe was followed spectrophotometrically at 310 nm. The activity of pepsins towards short synthetic substrates were investigated using ninhydrin. Inhibition studies were performed with the reversible inhibitor pepstatin A and the inhibitors DAN, EPNP and p-bromophenacyl bromide. Ostrich pepsins exhibited an optimum pH of 2.0 towards haemoglobin, and were stable up to pH 7. 5. The optimum temperature for ostrich pepsin II was at 60°C, while ostrich pepsin I showed a broader optimum temperature range (40-60°C). Ostrich pepsin I was more susceptible to heat inactivation than ostrich pepsin II. Both pepsins showed a lower activity towards haemoglobin than porcine pepsin. Only ostrich pepsin II could hydrolyse the hexapeptide substrate, albeit at a much slower rate than porcine pepsin. The activity of the ostrich pepsins towards short synthetic peptides was generally very low. Pepstatin A is a very potent inhibitor of ostrich pepsins with a K i of 2.14-2.2 × 10 -8 M. EPNP, DAN and p-bromophenacyl bromide were all inhibitory to ostrich pepsin It, DAN requiring the presence of Cu 2÷ . Ostrich pepsins exhibited similar thermal and kinetic properties when compared to pepsins of avian origin. Avian pepsins are generally more stable to alkaline conditions, and exhibit low activity towards short synthetic substrates. Keywords: Ostrich

Pepsins

Kinetic

pH

lnhibitors

Temperature

Int. J. Biochem. Cell Biol. (1995) 27, 1293 1302

INTRODUCTION

Pepsin A (EC 3.4.23.1 ) and gastricsin (pepsin C) (EC 3.4.23.3) are widely distributed enzymes. and have been detected in the gastric juices of birds, amphibia, fish and mammals. Pepsins as well as gastricsins are both capable of catalysing transpeptidations of both the amino- and carboxyl-transfer type (Ryle. 1988), with the primary site of action being a CO NH bond to which a L-tyrosyl or L-phenylalanyl contribute *To whom all correspondence should be addressed. dbbreriations: APDT. N-acetyl-L-phenylalanyl-3,5-diidoL-tyrosine: APT, N-acetyl-D-phenylalanyl-L-tyrosine; CBz, carbobenzox 5: DAN, diazoacetyI-DL-nonleucine methyl ester: EPNP. 1,2-epox~-3-(p-nitrophenoxy)propane. Received 9 June 1995: accepted I 1 July 1995. Bc 2" iz

L

to the NH group (Guerard and Le Gal, 1987). In the ostrich two forms of pepsinogens were purified, and activated to their respective pepsins by means of acidification (Pletschke et al., 1995). The present study was aimed at the kinetic and thermodynamic characterization of the two ostrich pepsins. MATERIALS AND METHODS

Materials Porcine pepsinogen and pepsin, bovine haemoglobin, N-Ac-DPhe-Tyr, N-CBz-GluYyr. N-CBz-Glu-Phe, N-CBz-GIy-Phe, N-CBzGly-Tyr, APDT~ DAN, EPNP and pbromophenacyl bromide were supplied by Sigma Chemical Co, U.S.A. Leu-Ser-PheNO),-Nle-Ala-Leu-OMe, CBz-His-Phe-Trp-

129~

1294

Brett I Pletschkeet al

OEt and CBz-His-Phe-Yyr-OEt were purchased from Bachem Feinchemikalien AG, Bubendorf, Switzerland. All other chemicals were of the best analytical grade available. Ostrich pepsinogens I and lI were isolated and purified by ammonium sulphate fractionation, Toyopearl Super Q-650S chromatography and rechromatography, and hydroxylapatite chromatography of a pH 8.0 mucosal extract. Pepsins were obtained by acidification of pepsinogens and were purified by chromatography on a SP-Sephadex C-50 column (Pletschke et al., 1995). Actit'itv determination with haemoglobin

Peptic activity was determined according to the method described previously (Pletschke et al., 1995). Activity determinations with synthetic suhstrates Hexapeptide substrate. The action of ostrich pepsins on the hexapeptide substrate was assayed according to the method of Martin (1984) as described by Guerard and Le Gal (1987). The enzymatic cleavage of the Phe(NO2)-Nle bond in Leu-Ser-Phe-(NOz)-Nle-Ala-Leu-OMe was followed spectrophotometrically at 295 or 310nm. The incubation mixture contained 500/~ 1 of the hexapeptide (0.175 mM 1 and 25 l~l pepsin solution (containing 0.5 l~g of porcine pepsin A or 25/xg of ostrich pepsin I or 1.25/xg of ostrich pepsin Ill in 0.1 M Na acetate buffer, pH 4.7. The absorbance changes at 310 nm were followed continuously at 3 0 C for at least 1 rain. Martin (1984) proposed that the hexapeptide hydrolysis can be followed by difference spectrophotometry at 310nm (pH 3.0 and above) and at 295 nm (pH 3.0 and lower). Di- and tripeptide substrates. Peptidase activity was determined using the substrates APDT, APT, N-CBz-Glu-Tyr, N-CBz-GIy-Tyr, N-CBz-GIu-Phe, N-CBz-GIy-Phe, CBz-HisPhe-Trp-OEt, and CBz-His-Phe-Tyr-OEt, according to a modification of the original method of Chiang et al. (1966), by Abuharfeel and Abuereish (1984). Activity was determined in a 200/~1 solution containing 0.5 mM substrate in 0.1 M Na citrate buffer, pH 2.0, for the dipeptide substrates and APDT/APT, and in 0.04 M Na citrate buffer, pH 4.0, for the N-CBz-HisPhe-Trp/Tyr-OEt substrates. The reaction mixture contained either 5/~g porcine pepsin A. 12.5/~g ostrich pepsin I or 25 #g ostrich pepsin II, and was maintained at 37 C for 20 rain,

followed by the addition of 200 Fal 2% ninhydrin solution. The reaction mixture was placed in a boiling water bath for 15 min, cooled in ice, and finally diluted with 2.0 ml 60% (v/v) ethanol. The absorbance at 570 nm was a measure of the extent of hydrolysis. All tests were run in duplicate, and duplicate blanks were performed by adding the substrate after enzymes were inactivated by the addition of the ninhydrin reagent. p H optimum and stability studies

In order to determine the optimum pH values, porcine pepsin A and ostrich pepsins I and II were assayed in 0.05 M HC1-Na acetate buffers in the pH range 1-5 as described by Moriyama et al. (1985), using haemoglobin and the hexapeptide as substrates. The alkaline stability of these enzymes as well as their corresponding pepsinogens were determined by dissolving aliquots of the pro-enzymes ( I 10 #g) in 50 #1 distilled water and adding to it 350/xl buffer (0.1 M) at pH 7.5 (Na phosphate) and pH 8-12 (Na borate). The alkaline reaction mixtures were kept at room temperature (20 + 1 C ) for 20 rain, and acidified to pH 2.0 with HCI. The concentration and volume of acid required was determined in a preliminary experiment. The remaining activity was assayed as before at 37~C for 10min with 1% haemoglobin as substrate, and expressed as a percentage of the maximal activity. The time course of alkaline inactivation of porcine and ostrich pepsins was determined by incubation of the pepsins in 0.1 M Na phosphate buffer, pH 8.0, at 0 C for 60min. Aliquots were withdrawn at 0, 5, 10, 15, 30, 40 and 60 rain, and tested for remaining proteolytic activity as described above, and expressed as a percentage of the maximal activity. Temperature studies

The optimum temperatures for porcine and ostrich pepsin activities were determined by incubating pepsins (1-10 iLg) in 450/xl 0.1 M Na citrate buffer, pH 2.0, at 10, 20, 30, 40, 50, 60 and 7 0 C with 750 #1 of 1% haemoglobin. After 10 min, the reaction was terminated by the addition of 1 ml 10% TCA. All determinations were done in duplicate, and appropriate controls were performed at each temperature. Remaining activity was expressed as a percentage of the maximal activity. The energy of activation (E,) was calculated from an Arrhenius plot using the temperature activity data from 10 to 6 0 C for porcine pepsin

1295

Ostrich pepsins l and II

A and ostrich pepsin II and 10 to 4 0 C for ostrich pepsin I. Linear regression analysis was performed and the free energy of activation (AG*) determined by a modification of the Arrhenius plot as described by Cornish-Bowden (1976) in which log (k/T) is plotted against I/T. The thermal stability of the pepsins was investigated by preincubation of the enzymes (1 10/~g) in 200/~1 0.1 M Na citrate buffer, pH 2.0, at 10, 20, 30, 40, 50, 60 and 7 0 C for a period of 1 hr. Aliquots of 50 # 1 were removed, and added to 450/~1 0.1 M Na citrate buffer, pH 2.0, and 750 I~1 1% haemoglobin. The mixtures were incubated at 37 C for 10 min and proteolytic activity determined as before. All tests and appropriate controls were performed in duplicate, and remaining activity was expressed as a percentage of maximal activity.

Kinetic parameters Michaelis-Menten coefficients (Kin) and maximum velocities (I/,~) were determined by least-square regression analysis of Lineweaver Burk, Hanes and Eadie Hofstee plots. Concentrations of the haemoglobin substrate varied from 0 to 0.5% (w/v) ((~77.5/~M) and (~300/~M for the hexapeptide substrate. The catalytic constants (k~,) and specificity constants (k~,~/K~) were determined for the pepsins as described by Fersht (1985).

Inhibition by pepstatin A and determination of K, Porcine pepsin A and ostrich pepsin II were assayed in the presence of increasing concentrations of pepstatin A (1:64 to 64:1 molar ratio of inhibitor:enzyme) with 1% haemoglobin (final concentration of haemoglobin was 0.625%) at pH 1.0, 2.0 and 3.0 for different time intervals up to 60rain. as described by Kageyama and Takahashi (1976). To determine the type of inhibition and inhibition constant (K~), assays were performed at increasing concentrations of inhibitor with two different haemoglobin concentrations: 0.25 and 1% lbr porcine pepsin A, 0.3 and 1% for ostrich pepsin I, and 0.5 and 1% for ostrich pepsin [I. The inhibitor constants (K,) were calculated by leastsquare regression analysis of Double-Dixon plots (Dixon and Webb, 1979).

Inhibitoo' effect of DAN, bromophenacyl bromMe

the method of Kageyama and Takahashi (1976). The pepsins (30 #g) were incubated with pbromophenacyl bromide (15, 62.5 and 500/~g), EPNP (625, 2500, 3750 and 5000/tg) and DAN (7.5 and 30/~g). DAN was also assayed in the presence or absence of Cu 2+ at the 30/~g level. The remaining proteolytic activity with haemoglobin as substrate was determined as before and expressed as a percentage of the maximal activity at zero time. RESULTS

pH optimum and stability studies The pH optima for ostrich pepsins I and II as well as for porcine pepsin A, with haemoglobin as substrate, were 2.0 in all three cases (Fig.l). All three pepsins showed similar pH dependency profiles, although ostrich pepsin I exhibited increased activities over a broader pH range (2 4) as compared to the two other pepsins. Porcine pepsin appeared to be most sensitive to non-optimal pH values, especially the higher pH values. The pH optimum of ostrich pepsin II with the hexapeptide substrate was found to occur over a broad pH range from 3.5 to 5.2, whereas that of porcine pepsin was found to be around 4.2 (Fig.2). No activity with the hexapeptide was detected for ostrich pepsin I. The alkaline stability of ostrich and porcine pepsinogens and pepsins is shown in Fig.3. Ostrich pepsins I and II were shown to be stable up to pH 7.6, and were rapidly inactivated above pH 8.6. Porcine pepsin A was found to be inactivated above pH 6.0. Ostrich pepsinogen I and porcine pepsinogen were shown to be stable up to pH 9.5 and were rapidly inactivated above pH 10.6 in the case of porcine pepsinogen, and 120

-~ so ~

t

60

.,

'

z < 40

EPNP and p-

The reaction of DAN, EPNP and pbromophenacyl bromide with porcine pepsin and ostrich pepsin II was examined according to

0 0

I

2

3

4

5

pH

Fig 1. pH dependence of activities of ostrich pepsin isozymes and porcine pepsin on haemoglobin. (B), porcine pepsin: (11~'). ostrich pepsin ] and ( + L ostrich pepsin It.

1296

Bren I. Pletschke et al. c 20

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2 20

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35

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5

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6

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pH

C

Fig. 2. pH dependence o f activities of ostrich pepsin 1[ and porcine pepsin on Leu-Ser-Phe-(NO:i-Nle-Ala-Leu-OMe. ( × ), porcine pepsin and (m), ostrich pepsin 11.

above pH I 1.1 for ostrich pepsinogen I. Ostrich pepsinogen II was more stable under alkaline conditions and was completely inactivated only above pH 12.1. Ostrich pepsins showed a remarkable alkaline stability at pH 8.0, while porcine pepsin rapidly lost activity after 5 rain at this pH (Fig.4).

Temperature studies

The temperature profile for ostrich pepsin II was found to be similar to that of porcine pepsin (Fig.5). The optimum temperature for both these pepsins was found to be 6OC, after which a rapid decrease in activity was noticed. Ostrich pepsin I, however, exhibited a wider range of optimum activity, from 40 to 60' C, but it also rapidly lost activity above 60 C. Ostrich pepsin II also exhibited a temperature stability profile

,

1C

20

30

INCUBATION

40 50 T I M E (MIN)

Fig. 4. Alkaline stability of ostrich and pH 8.0. Pepsins were dissolved in 0.1 buffer, pH 8.0, and were maintained Aliquots of 50#1 were withdrawn at intervals, and the remaining activity (m), porcine pepsin; (jlt-), ostrich pepsin pepsin II.

60

70

porcine pepsins at M Na phosphate at 0'~C for 1 hr. appropriate time was determined. I and ( + ) , ostrich

similar to that of porcine pepsin (Fig.6), with both pepsins being stable at temperatures up to 5 0 C for 1 hr. Compared to the latter two pepsins, ostrich pepsin I appeared to be more temperature sensitive, with its activity declining steadily as the temperature was increased. From Arrhenius plots the energies of activation (E,) for ostrich pepsins I and II, and porcine pepsin were determined: 38.9, 43.5 and 46.6 kJ.mol ~, respectively. AG values for ostrich pepsins I and lI, and porcine pepsin were 81.2, 77.8 and 79.9 kJ.mol ~, respectively. Kinetic' p a r a m e t e r s

Kinetic parameters for porcine and ostrich pepsins are presented in Table 1. An inherent

120

100 ' ,, 1t0 100 '

80

90 60

80

4O

60

70

50 2O

40

L 0-~ 75

6

30 95

105

11 5

12 5

20

pH 10

Fig. 3. Alkaline stability of ostrich and porcine pepsinogens and pepsins. Pepsins and pepsinogens (1 10#g in 50ttl distilled H:O) were incubated in 350#1 buffer (Na phosphate at pH 7.5 and Na borate at pH 8 12) at 20.5 C for 20 min. Before assaying, the alkaline reaction mixtures were acidified to pH 2.0 by the addition of HC1. ( . ) . porcine pepsin: ('~lr), ostrich pepsin 1; ( + ) . ostrich pepsin lI: ([Z), porcine pepsinogen: (,~), ostrich pepsinogen I and ( x ). ostrich pepsinogen II.

0" 5

15

25

35 45 55 TEMPERATURE (°C)

65

75

Fig. 5. Influence of assay temperature on ostrich and porcine pepsin activity. Haemoglobin solution (750#1 1% haemoglobin) was incubated at different temperatures with 450 #1 pepsin solution (1 10#g) for 10rain. Remaining activity was determined. (m), porcine pepsin; ( ~ ) , ostrich pepsin I and ( + ) , ostrich pepsin lI.

Ostrich pepsins 1 and I1

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TEMPERATURE

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Fig. 6. Thermal stability or ostrich and porcine pepsins. Pepsins (I l0 ~g) were dissolved in 50 ,al distilled water and incubated at different temperatures for l hr. Remaining activity was determined at 37 C after the addition of 450 pl 0.1 M Na citrate buffer, pH 2.0, and 7501~1 haemoglobin. ( i ) , porcine pepsin: ("k), ostrich pepsin 1 and ( + ), ostrich pepsin 11.

problem exists in the d e t e r m i n a t i o n of the catalytic a n d specificity constants for protein substrates such as h a e m o g l o b i n , using the method of A n s o n (1938). Some authors have adopted Vmax/Km,ap p tO describe physiological efficiency, as will be discussed below. Ostrich pepsin I revealed a lower K,,.,pp value with h a e m o g l o b i n than the porcine enzyme, whereas ostrich pepsin II showed approximately a 7-fold increased Km.~pp value when c o m p a r e d to pepsin I. W h e n c o m p a r e d to porcine pepsin, both ostrich pepsins showed a 7 to 26-fold lower V~,~ value. The l/ma×/Km.,pp values for ostrich pepsins were only 9 16% of that of porcine pepsin, indicating that both ostrich pepsins are not as efficient as porcine pepsin in hydrolysing haemoglobin. The hexapeptide substrate was also hydrolysed more efficiently by porcine pepsin than by ostrich pepsin ii (k~, KI~, = 356.41 vs 135.71 sec ~.mM ~, respectively). The effect of pH on the hydrolysis of the hexapeptide sub-

}-ig. 7. The effect of pH on the specificityconstant of ostrich and porcine pepsin-catalysed hydrolysis of the hexapeptide substrate. ( i ) , porcine pepsin and ( x ), ostrich pepsin II.

strate can be seen in Fig.7. The activity o f ostrich pepsin II was d e p e n d e n t on the ionization of two groups whose p K , values are 2.4 and 4.25. F o r porcine pepsin, p K , values of 2.4 and 5.4 were found. Ostrich pepsin II showed a 2.4-fold lower Kn, value with the hexapeptide subs+rate when c o m p a r e d to porcine pepsin. Ostrich pepsin 11 was also less efficient in hydrolysing A P D T ; the kc,, value of ostrich pepsin II was 9.5 times lower than that of porcine pepsin, resulting in a very low specificity constant of 0.02sec ~-mM ~. Ostrich pepsin I showed no activity with the hexapeptide substrate or A P D T . Ostrich pepsin I displays properties similar to those of m a m m a l i a n gastricins (compare their inability to hydrolyse A P D T as substrate) (Tang, 1970). The specific activities of porcine a n d ostrich pepsins with various synthetic substrates were investigated (Table 2). Generally, the specific activities of ostrich pepsins with synthetic substrates were rather low. Porcine pepsin hydrolysed A P D T a n d C B z - H i s - P h e - T r p - O E t quite well, and also showed some activity with N-CBz-GIy-Phe and CBz-His-Phe-Tyr-OEt.

[able 1 Kinetic parameters for ostrich and porcine pepsins Substrale Haemoglobin+ Hexapeptide: Leu-Ser-Phe-(NO,)Nle-Ala-Leu-OMe; APDT+

Enz}I1]e

Porcine pepsin Ostrich pepsin I Ostrich pepsin II Porcine pepsin Ostrichpepsin 11

I/m,,,*

Km

(raM)

12 668 480 1776

Km,,pp

(HM) 9.94 "~ "~ ,.4~ [6.06

0.167 0.0711

kcat (sec ~)

59.52 9.5

kcat/Kin (sec I ' / / M i)

--356.41 135.71

Porcine pepsin 0.057 0.019 0.33 Ostrich pepsin I1 0.100 0.002 0.02 *Due to the nature of the haemoglobin assay I',..... values are AA,~0,,,,,.hr ~.mg enzyme and AA:so~m'hr +'rag enzyme ~'~m substrate +Assay temperature and pH 3"7 C and 2.0. respectively. +Assay temperature and pH 30 C and 4.7. respectively.

[/ma×/,Km,app 1247.45 197.53 I10.59

---

Vma~,Km,,,ppvalues are

Bren 1. Pletschke et al.

1298

Table 2. Specific activities of porcine and ostrich pepsins with various synthetic substrates Substrate N-Ac-D-Phe-Tyr N-CBz-GIu-Tyr N-CBz-Gly-Phe N-CBz-GIu-Phe N-CBz-Gly-Tyr APDT Cgz-His-Phe-Tyr-OEt CBz-His-Phe-Trp-OEt

Porcine

OstrichI

OstrichII

0 0.93 4.77 1.63 0 17.33 9.8 16

2.33 0 1.67 0 1.33 0 0 0

0.24 0.28 0.02 0 0.55 2.17 0 2.2

For all substrates, specific activib, units are AAs;0.m.20rain ~.mgprotein ~. Assays ,,,ere performed at 37 C and pH 2.0, except for the substrates CBz-His-Phe-Tyr-OEt and CBz-His-Phe-Trp-OEt (pH 4.0).

Ostrich pepsins showed no activity with N-CBzG l u - P h e and C B z - H i s - P h e - T y r - O E t . while porcine pepsin was inactive with N - A c - D - P h e T y r a n d N-CBz-GIy-Tyr.

Inhibition by pepstatin A and the determination of K, Figure 8 depicts the effect of increasing molar ratios of pepstatin A on porcine pepsin and ostrich pepsins 1 a n d II at pH 2.0. I n h i b i t i o n was less p r o n o u n c e d at pH 1.0 and 3.0 (results not shown). I n h i b i t i o n was fairly rapid and a time period of 10 m i n was required t'or inhibition. Ostrich pepsin I required inhibitor to enzyme levels above 1 : 1 (between 1 : 1 and 4:1 ) for 50% i n h i b i t i o n of activity, whereas ostrich pepsin II required levels between 1 : 4 and I : 1 for 50% inhibition. Porcine pepsin A required a m o l a r ratio of inhibitor : enzyme of approx. 1 : 4 for the same effect. K, values, obtained from D o u b l e - D i x o n plots (figures not shown), were

110

-

2 . 1 4 x 10 s, 2 . 2 x l0 -~ and 1 x 10 9 M for ostrich pepsins I a n d II a n d porcine pepsin, respectively and competitive i n h i b i t i o n was observed for all three enzymes.

Effect 0/ DAN, EPNP and p-bromophenacyl bromide The effects of D A N , E P N P a n d p b r o m o p h e n a c y l b r o m i d e on porcine pepsin a n d ostrich pepsin II activities are shown in Figs 9-1 I, respectively. The presence o f C u 2+ in the form of cupric sulphate appears to be essential for D A N to react with the pepsins. A level of 30/~g inhibitor was required to completely inhibit the pepsin activity. However, in the absence of 400/~M CuSO4, this level of inhibitor could not exert its effect. In the presence of 1!0

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20

50

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PEPSTATIN/ENZYME

(MOL/MOL)

Fig. 8. Inhibition of ostrich and porcine pepsins by pepstatin. Pepsins were dissolved in 2.5 ml 0.1 M glycine HCI buffer, pH 2.0. and incubated with various amounts of pepstatin dissolved in 2.5 ml 0. I M glycine HC1 buffer, pH 2.0. After incubation at 37C for 10min. 400/J1 aliquots were withdrawn, 750t, 1 of 1% haemoglobin added, and assayed for remaining activity at 37C. (73), porcine pepsin: (O). ostrich pepsin 1 and (O), ostrich pepsin I1

°0

1

2

3 REACTION

4 TIME

5

6

(hr)

Fig. 9. Inhibition of ostrich and porcine pepsins by DAN. Aliquots were withdrawn at appropriate time intervals and the remaining activity was determined. Pepsin (30/~g) and various amounts of DAN were incubated in 4 ml of 0.05 M Na phosphate buffer, pH 6.0, at 25 C. (E]), porcine pepsin with 30/~g DAN in the absence ofCu2+; ('~), ostrich pepsin It with 30/~g DAN in the absence of Cu2+; (+), porcine pepsin with 7.5/,tg DAN in the presence of 400/, M CuSO4; ( x ), ostrich pepsin II with 7.5,tg DAN in the presence of 400 # M CuSO4; ( I ) , porcine pepsin with 30 ,ug DAN in the presence of 400,uM CuSO4; (A), ostrich pepsin II with 30#g DAN in the presence of 400#M CuSO4,

1299

Ostrich pepsins 1 and 1I ~1o

Cu 2+, 1 hr was sufficient for D A N to fully exert its action. EPNP required a much longer time period of 42 hr to exert its maximal effect on the pepsins (Fig. 10). A level of 5000#g per assay was needed to inhibit ostrich pepsin I1 and porcine pepsin to 4.9 and 0% of maximal activity respectively, while 625 Fg per assay was insufficient to inhibit pepsin activity at all. Varying concentrations of p-bromophenacyl bromide (15 500 #g) led to relatively small inhibitory effects, especially in the case of porcine pepsin. More substantial effects were, however, evident in the case of ostrich pepsin II. The action of p-bromophenacyl bromide on pepsins was very slow, and after 67 hr, the porcine pepsin was inhibited to between 44 and 54% of maximal activity. The remaining ostrich pepsin II activity after 67 hr was down to 25 30% of maximal level (Fig. 11).

lOO~ 90 8O

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60 5o

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30 20 10

%

27 REACTION

48 TIME

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---72

(hr)

Fig II. Inhibition of ostrich and porcine pepsins by p-bromophenacyl bromide. Aliquots were withdrawn at appropriate time intervals and the remaining activity was determined. Pepsin (30 #g) and various amounts of p-bromophenacyl bromide (pBPB) were incubated in 4 ml 0.05 M NazHPO4-citrate buffer, pH 2.8, at 25C. (m), porcine pepsin with 15 l*g pBPB; (A), porcine pepsin with 62.5/,zg pBPB: (+ l, porcine pepsin with 500/~g pBPB; ('~), ostrich pepsin II with 15Fg pBPB: ([B), ostrich pepsin II with 62.5 l*g pBPB and ( × ), ostrich pepsin I1 with 500 Fg pBPB.

DISCUSSION Ostrich pepsins I and II, as well as many other pepsins documented in the literature optimally cleave haemoglobin at pH 2.0 (Fig. 1). With denaturation, the optimal activity of pepsin may be displaced from pH 2.0 to 3.5 (Schlamowitz and Peterson, 1959). Pepsin is a protease of broad side-chain specificity, but in general, the bonds most susceptible to proteolytic attack are dipeptidyl units containing at least one hydrophobic amino acid residue such as Phe, Tyr, Leu and Met (Fruton, 1987). Esumi et al. (1980) .lOT ~0o~

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60

Z

50 ! 40 i

e£

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30' 20,

00

24 REACTION

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~S

T I M E (hr)

Fig. 10. Inhibition of ostrich and porcine pepsins by EPNP. Aliquots were withdrawn at appropriate time intervals and the remaining activity was determined, Pepsin (30/xg) and various amounts of EPNP were incubated in 4 ml 0.05 M Na citrate buffer, pH 4.6, at 25 C (Ill). porcine pepsin with 5000/*g EPNP; (•), ostrich pepsin 1[ with 5000/~g EPNP: ( t ), porcine pepsin with 2500 pg EPNP: ( x ], ostrich pepsin II with 3750#g EPNP: ([]L porcine pepsin with 6251~g EPNP: and ("kL ostrich pepsin I1 v~ith 625 t~g EPNP.

have reported pH optima for quail and chicken pepsins of 3,0. Streicher et al. (1985) have also reported a pH optimum with haemoglobin of 3.6 for partially purified ostrich pepsins, which is higher than the pH optimum observed in the present study. Most investigators have conducted their studies on the hexapeptide Leu-Ser-Phe(NO2)Nle-Ala-Leu-OMe at pH 4.7, to allow for comparison with other aspartic proteases in the literature (Martin, 1984; Guerard and Le Gal, 1987). In the present study both porcine and ostrich pepsin lI revealed a substantial amount of activity at pH 4.7 (Fig. 2). However, one interesting but complicating aspect of the hexapeptide assay is the reported dependence of the spectral properties of the chromophoric tripeptide Leu-Ser-Phe-(NO2), which is split off by the action of pepsin, on pH. The chromophoric tripeptide was found to have a continuously changing extinction coefficient with a change in pH at 295 and 310 nm (Martin, 1984). From Fig. 7 the pK, values of catalytic residues for ostrich pepsin [I (2.4 and 4.25) and porcine pepsin (2.4 and 5.4) are different from those obtained for bovine pepsin A by Martin (1984), who obtained values of 1.2 and 5.0. These p K , values strongly suggest the involvement of two carboxylic acids, probably Asp-32 and Asp-215, in the active site (Martin, 1984). Generally speaking, pepsinogens from mammalian, teleost or avian origin are stable under

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BretI I Pletschke et al.

moderately alkaline conditions, whereas pepsins are denatured rapidly at pH values above 6 (Foltmann, 1981). Porcine pepsin A was found to be stable up to pH 6.0, whereas chicken pepsin was shown to be stable up to pH 7.5, indicating species differences in alkaline stability of the enzyme (Foltmann, 1981). The present study would seem to indicate that ostrich pepsins/pepsinogens resemble those from other avian species, in that they are more resistant to alkaline denaturation than their mammalian counterparts (Figs 3 and 4). Bohak (1969) reported that the alkaline stabilities of chicken and porcine pepsinogens were essentially similar. Chicken pepsin was found to be stable up to pH 8.0 under similar conditions, whereas porcine pepsin was rapidly inactivated at pH 7.0. Japanese quail pepsin was found to be stable up to pH 8.5 (Esumi et al., 1980), while duck pepsin was stable at pH 7.5, and was completely inactivated at pH 9.6 (Pichova and Kostka, 1990). Duck pepsinogen was reported to be stable at pH 10 and 25C~ but was completely inactivated at pH 12.1 (Pichovfi and Kostka. 1990). McPhie (1975) reported that unfolded pepsinogen at pH 8.5 can be renatured at pH 7.0. Intermediate exposure to neutral pH seems to return the protein to a form which can be activated. Optimum temperature profiles for ostrich pepsin II and porcine pepsin were identical, with ostrich pepsin I exhibiting optimum activity over a wider temperature range (Fig. 5). Ostrich pepsin II also exhibited similar thermal stability as porcine pepsin (Fig. 6), indicating that the thermal properties of ostrich pepsin II resemble those of other homeotherms. Ostrich pepsin I was, however, more susceptible to inactivation by heat (Fig.6), which is reminiscent of gastric proteases present in poikilothermic animals (Squires et al., 1985a,b; Arunchalam and Haard, 1985; Gildberg and Raa, 1983). Arrhenius activation energies for ostrich and porcine pepsins in the present study were high (38.9~46.6kJ.mol l) when compared to those of poikilothermic origin (13.4 and 12.1 kJ" mol ' for polar cod pepsinogens A and B, respectively) as described by Aruchalam and Haard (1985). Ostrich pepsin II, unlike ostrich pepsin I, appears to be rather similar to porcine pepsin in terms of pH and temperature optima. thermal stability and hydrolysis of the hexapeptide substrate.

Ostrich pepsins I and II were not as efficient as porcine pepsin in hydrolysing haemoglobin as a substrate (Table 1). Vundla et al. (1992) have proposed that higher Km values may indicate a physiological function, such as a slower digestive process. The low Km value described for ostrich pepsin I may be linked to the rapid digestive processes present in all avian species, including the ostrich. It must be remembered, however, that Km values reported for haemoglobin are not true kinetic constants, as each value represents the average for the hydrolysis of several bonds occurring simultaneously at different velocities (Vundla et al., 1992). It must also be remembered that the Km values are dependent on the duration of the digestion and other experimental conditions. Porcine pepsin was much more efficient than ostrich pepsin II in hydrolysing the hexapeptide substrate and APDT (Table 1). Pepsins of avian, amphibian and teleost origin generally exhibit a very low degree of activity towards shorter synthetic peptide substrates (Esumi et al., 1980; Bohak, 1969; Ward et al., 1978; SS~nchez-Chiang et al., 1987; Donta and Van Vunakis, 1970). This low level of activity may be related to the length of the substrate involved. Extending the length of the peptide chain of a substrate towards either the amino or carboxyl terminus greatly increases the k~,~ value, with relatively little variation in Km. Aspartic proteases such as pepsin have an extended binding cleft capable of accommodating peptides of up to seven residues. As substrates become larger, more of the binding energy could be used to lower the activation energy of hydrolysis, resulting in enhanced kca, values. Mammalian pepsins such as porcine pepsin, possess extended binding clefts more adept at interacting with the synthetic substrate, forcing distortion of the substrate peptide bond from planarity and opening of the substrate for nucleophilic attack (Foltmann, 1981; Pearl, 1987; James and Sielecki, 1987). Synthetic substrates such as CBz-His-PheTyr-OEt and CBz-His-Phe-Trp-OEt are cationic, and exhibit a pH optimum of about 4, compared to 2 3 for the acyl dipeptides (such as APDT and the other dipeptides used in this study). This is a consequence of the fact that the carboxylate ion in acyl dipeptides adjacent to the sensitive bond is strongly inhibitory to pepsin action above pH 2.0 (due to the state of ionization of the carboxylate ion) (Fruton, 1970, 1987).

Ostrich pepsins 1 and II P e p s t a t i n A was a very p o t e n t c o m p e t i t i v e i n h i b i t o r o f o s t r i c h a n d p o r c i n e pepsins (Fig. 8). O s t r i c h p e p s i n s r e v e a l e d K, v a l u e s o f 2.142.2 x 10 ~ M, a 20-fold h i g h e r v a l u e t h a n that for p o r c i n e pepsin. A K, v a l u e for d u c k p e p s i n w i t h the s y n t h e t i c s u b s t r a t e p y r o G l u - H i s - P h e ( 4 N O 2 ) - P h e - A I a - L e u N H : was r e p o r t e d to be a p p r o x . 1 × l0 ~°M (Pichovfi a n d K o s t k a , 1990). R y / e ([988) has r e p o r t e d Ki v a l u e s o f I x 1 0 - g M for the b i n d i n g o f p e p s t a t i n to pepsins, while F e r s h t (1985) r e p o r t e d a K, for p e p s t a f i n w i t h p o r c i n e pepsin o f 4.5 × 10 ~ M. Like the m a j o r i t y o f a s p a r t i c p r o t e a s e s , ostrich pepsin II was i n h i b i t e d by D A N , E P N P a n d p - b r o m o p h e n a c y l b r o m i d e . I n h i b i t i o n ot" o s t r i c h pepsin lI o c c u r r e d r a t h e r r a p i d l y with D A N (1 h r was required), a n d the p r e s e n c e o f C u 2+ was essential for i n h i b i t i o n (Fig. 9). T h e r e q u i r e m e n t o f D A N for C u 2+ is well d o c u m e n t e d (Stein, I970, K a g e y a m a a n d T a k a h a s h i ,

1976, 1980, 1984: Athauda et al., 1989; Tanji et al., 1988; Kageyama et al., 1983). EPNP and

p-bromophenacyl bromide required a longer period of time (42-67 hr) to inhibit the pepsins (Figs 10 and l l, respectively). This extended period of time required by EPNP and p-bromophenacyl bromide for inhibition has also been reported by other investigators (Kageyama and Takahashi, 1976: 1980, 1984; Athauda et al., 1989; Tanji et al., 1988; Kageyama et al., 1983). In conclusion ostrich pepsins appear to exhibit similar pH stability, pH optima, and thermal and kinetic properties when compared to other pepsins of avian origin. Generally speaking, avian pepsins are more stable to alkaline conditions, and exhibit rather low specific activities towards short synthetic substrates. Acknowledgements--The authors gratefully acknowledge financial support provided by the Foundation for Research Development and the University of Port Elizabeth. We also express our gratitude to the Klein Karoo Agricultural Co-operation at Oudtshoorn, South Africa, for their generous supply of experimental material.

REFERENCES

Abuharfeel N. M. and Abuereish G. M. 1984) Isolation and characterization of came] pepsins. C~#np. Biockem. Ph.vsiol. 77A, 175- 182. Anson M. L. (19381 The estimation of pepsin, trypsin. papain and cathepsin with hemoglobin. J. Get?. Phyvio/ 22, 79-89. Arunchalam K. and Haard N. E (]985) Isolation and characterization of pepsin from polar cod (Boreogadus saida) Comp. Biochem. Phvsio/ 80B, 467 473.

1301

Athauda S. B. P., Tanji M. and Takahashi K. (1989) A comparative study on the NH,-termina~ amino acid sequences and some other properties of six isozymic lbrms of human pepsinogens and pepsins. J. Biochem. 106, 920 927. Bohak Z. (19691 Purification and characterization of chicken pepsinogen and chicken pepsin. J. Biol. Chem. 244, 4638-4648. Chiang L,. Sanchez-Chiang L., Wolf S. and Tang J. (1966) The separate determination of human pepsin and gastricsin. Proc. Soc. Exp. Biol. Med. 122, 700 704. Cornish-Bowden A. (1976) Prineiples ol° Enzyme Kinetics, pp. 1 I I. Butterworths, London. Dixon M. and Webb E. C. (1979) Enaymes, 3rd Edn, pp. 344-353. Longman, London. Donta S. T. and Van Vunakis H. (1970) Chicken pepsinogens and pepsins. Their isolation and properties. Biochemistry 9, 2791 2797. Esumi H., Yasugi S., Mizuno T. and Fujiki H. (1980) Purification and characterization of a pepsinogen and its pepsin from proventriculus of the Japanese quail. Biochim. Biophys. Acta 611, 363.-370. Fersht A. (t985) Enzyme Structure and Mechanism, 2rid Edn, pp. I03-106 and 4224.26. W. H. Freeman and Company, New York. f:ollmann B. (1981 ) Gastric proteases--structure, functions, evolution and mechanism of action. Es.vays Biochem. 17, 52 84. Fruton J. S. (1970) The specificity and mechanism of pepsin action. Adr. Enzymol. 33, 401 443. Fruton J, S. (1987) Aspartyl proteinases. In Hydrolytic [:?Izymes (Edited by Neuberger A. and Brocklehurst K.), pp . l 37. Elsevier Science Publishers B.V. (Biochemical Division) Inc.. New York. Gildberg A. and Raa J. (1983) Purification and characterization of pepsins from the arctic fish capelin (Mai/otu.~ ri[[osus1. Comp. Biochem. Physiol. 75A, 337 342. Guerard F and Le Gal Y. (1987) Characterization of a chymosin-Iike pepsin from the dogfish Scyliorhinus ccmicula. Comp. Biochem. Physiol. 88B, 823-827. James M. N G. and Sielecki A. R. (1987) Aspartic proteinases and their catalytic pathway. In Biological Macromolecules and Assemblies. Vol 3. Active Sites of Enzymes (Edited by Jumak F. A. and McPherson A). pp. 4134.82. John Wiley and Sons~ New York. Kageyama T. and Takahaslai K. (1976) Pepsinogens and pepsins from gastric mucosa of Japanese monkey. Purification and characterization. J. Biochem. 79, 455468. Kageyama T. and Takahashi K. (1980) Monkey pepsinogens and pepsins: V. Purification, characterization and amino-terminal sequence. Determination of crab-eating monkey pepsinogens and pepsins. J. Biochem. 88, 635 -645. Kageyama T. and Takahashi K. (1984) Rabbit pepsinogens: purification, characterization, analysis of the conversion process to pepsin and determination of the NH 2terminal amino-acid sequences. Fur. J. Biochem. 141, 261 269. Kageyama T., Moriyama A. and Takahashi K. (1983) Purification and characterization of pepsinogens and pepsins from Asiatic Black bear, and amino acid sequence determination of the NH~-terminal 60 residues of the major pepsinogen. J. Biochem. 94, 1557 1567.

1302

Brett I Pletschke et al,

Martin P. (1984) Hydrolysis of the synthetic chromophoric hexapeptide Leu-Ser-Phe-(NO2)-Nle-Ala-Leu-OMe catalyzed by bovine pepsin A. Dependence of pH and effect of enzyme phosphorylation level. Biochem. Biophvs. Acta 791, 28 36. McPhie P. (1975) The origin of the alkaline inactivation of pepsinogen. Biochemisto' 14, 5253 5256. Moriyama A., Kageyama T, Takahashi K. and Sasaki M (1985) Purification of Japanese Monkey prostate acid protease zymogen and its identification as a pepsinogen C-like zymogen. J. Biochem. 98, 1255 1261. Pearl L. H. (1987) The catalytic mechanism of aspartic proteinases. FEBS Lett. 214, 8 12. Pichov~i 1. and Kostka V. (1990) Molecular characteristics of pepsinogen and pepsin from duck glandular stomach. Comp. Biochem. Physiol. 97B, 89-94. Pletschke B. 1., Naud6 R. J., Oelofsen W., Muramoto K. and Yamauchi F. (1995) Ostrich pepsinogens I and 1I: purification, activation and chemical and immunochemical characterization of the enzymes from the proventriculus Int. J. Biochem. Cell. Biol. 27, 613 -624. Ryle A. P. (1988) Pepsins, gastricsins and their zymogens In Methods q/' Enzymatic Analysis. Volume V. En:ymes 3' Peptidases, Proteinases and their lnhibitors (Edited b'~ Bergmeyer H. U.. Bergmeyer J. and GraBl M.), 3rd edn, pp. 223 238. VCH Verlagsgesellschaft, Weinheim. Germany. SS_nchez-Chiang L., Cisternas E. and Ponce O. (1987) Partial purification of pepsins from adult and juvenile salmon fish Oncorhynchus keta. Effect of NaC1 on proteolytic activities. Comp. Biochem. Physiol. 87B, 793 797. Schlamowitz M. and Peterson L. U. (1959) Studies on the

optimum pH for the action of pepsin on "native" and denatured bovine serum albumin and bovine hemoglobin. J. Biol. Chem. 234, 3137-3145. Squires E. J., Haard N. F. and Feltham L. A. W. (1985a) Gastric proteases of the Greenland cod Gadus ogac. I. Isolation and kinetic properties. Biochem. Cell Biol. 64, 205 214. Squires E. J., Haard N. F. and Feltham L. A. W. (1985b) Gastric proteases of the Greenland cod Gadus ogac. II. Structural properties. Biochem. Cell Biol. 64, 215-222. Stein W. H. (1970) Chemical studies on purified pepsin. In Structure Function Relationships o f Proteolytic Enzymes (Edited by Desnuelle P., Neurath H. and Ottesen M.), pp. 253 260. Munksgaard, Copenhagen, Denmark. Streicher E., Naud6 R. J. and Oelofsen W. (1985) The isolation and characterization of pepsinogens from the proventriculus of the ostrich Struthio camelus. Comp. Biochem. Physiol. 82B, 67-72. Tang J. (1970) Gastricsin and pepsin. Meth. Enzymol. 19, 406 421. Tanji M., Kageyama T. and Takahashi K. (1988) Tuna pepsinogens and pepsins. Purification, characterization and amino-terminal sequences. Eur. J. Biochem. 177, 251 259. Vundla W. R. M., Brossard M., Pearson D. J. and Labongo V. L. (1992) Characterization of aspartic proteinases from the gut of the tick, Rhipicephalus appendiculatus neuman. Insect Biochem. Molec. Biol. 22, 405410. Ward P. H., Neuman V. K. and Chiang L. (1978) Partial characterization of pepsins and gastricsins and their zymogens from human and toad gastric mucosae. Comp. Biochem. Physiol. 61B, 491~.98.