THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 23, Ia%ue of December LO, pp. 12449-12455,1981 Printed in U.S.A.

The Mechanism of Trypsin Catalysis at Low pH PROPOSAL FOR A STRUCTURAL MODEL*

(Received for publication, April 28, 1981)

Eraldo Antoninil and Paolo Ascenzi From the Institute of Chemistry, Faculty of Medicine, University of Rome and Consiglio Nazionale delle Ricerche, Center for Molecular Biology, Rome, Italy

The steady state and pre-steady state kinetics of the trypsin-catalyzed hydrolysis of a-benzyloxycarbonyl- L-lysine-p-nitrophenyl ester (ZLNPE) have been stud- ied at different pH values from 2.5 to 8.0. The results are consistent with a three-step mechanism involving an acyl-enzyme intermediate

k+x k+a k+, E + S i= E * S e E * P + P l - E + P z .

k- I k-2 7G Evidence is presented for the formation of E = S being a pseudo-equilibrium process with a minimum value for the rate constant for the formation of E - S complex of -2 X 10’ M“ S-* .

The pH dependences have been determined for the dissociation constant of the E - S complex (Kb), as well as for the individual rate constants associated with the catalytic steps (k+2 and k+3). The pH profile of k+a/K,(= kCat/Km) reflects the ionization of groups of pK, value 4.5 +- 0.1, and 7.1 k 0.1 in the free enzyme (T = 21 k 1 “C, Z = 0.1). The pH dependence of k+2 implicates two ionizing groups in the acylation step with p& 3.75 f 0.1 and 7.1 0.1. The change in the value of K. with pH indicates a change in pK, of a group on binding the substrate. The pH dependence of k+3 implicates only one ionizing group in the catalytic step of pK, 7.1 f 0.1.

The pH dependence of the kinetic parameters shows that at acid pH values (2.55-4.80), the k,, step is rate- limiting in catalysis, whereas for pH values higher than 4.80, k+z becomes rate-limiting.

The change in K. and in the rate-limiting step with pH may be related to the acid-base equilibrium of Asp 177 present in the recognition site of trypsin.

Steady state and pre-steady state parameters for the trypsin-catalyzed hydrolysis of the noncationic sub- strate Q - benzyloxycarbonyl - L - alanine - p - nitrophenyl ester (ZANPE) in the presence and absence of ethylam- monium ion have been studied in the pH range between 3.0 and 8.0. In the absence of ethylammonium ion the pH dependence of kinetic parameters for the trypsin- catalyzed hydrolysis of ZANPE shows that the k+z step is rate-limiting in catalysis over the whole pH range explored. The presence of ethylammonium ion induced changes in the rate-limiting step with pH similar to those reported for ZLNPE. In addition, the affinity con- stant for the reaction of ethylammonium ion with tryp- sin changes with pH, and implies a pK, shift from 4.55 k 0.1 in the free enzyme to 3.75 2 0.1 in the enzyme-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed.

ethylammonium complex similar to that observed with ZLNPE.

For comparison, steady state and pre-steady state parameters for the a-chymotrypsin catalyzed hydroly- sis of ZLNPE and ZANPE have been obtained, The pH- independence (pH 3-6) of the rate-limiting step for a- chymotrypsin-catalyzed hydrolysis of ZLNPE and ZANPE (k+3 >> k+2) may be related to structural differ- ences observed at the active site of two proteases, where the neutral serine 189 in a-chymotrypsin has become the negatively-charged aspartic acid 177 in trypsin. Accordingly, no effects of ethylamine have been observed in the a-chymotrypsin catalyzed-hydrol- ysis of ZANPE.

The present communication is part of a continuing study of the detailed kinetics of reactions catalyzed by serine proteases (1, 2).

In a previous paper (2) it was demonstrated that cx-benzy- loxycarbonyl-L-lysine-p-nitrophenyl ester is an excellent sub- strate of trypsin, based on its kCat(limiting) = 145 s-l, the fastest known rate of trypsin catalysis. The overall pH profiles of K, and kcnt for the trypsin-catalyzed hydrolysis of ZLNPE’ are similar to those reported for benzoyl-DL-arginine-p-ni- troanilide (3), benzoyl-L-arginine-p-nitroanilide (4), tosyl-L- arginine methyl ester (5), and benzoyl-L-arginine ethyl ester (6).

The trypsin-catalyzed hydrolysis of ZLNPE closely con- forms to the classical mechanism involving a minimum of two intermediates:

k+l k+2 k+B

k-1 k-z k-3 E + S E.S * E.P - E + P z A

-F P1

SCHEME I

where PI isp-nitrophenol, P2 is a-benzyloxycarbonyl-L-lysine and E - P is considered to be an ester formed between the acyl moiety of the substrate and the serine hydroxyl group (Ser- 183) involved in the catalytic site of trypsin {7-11).

Earlier results (2) indicated that there is a change in the rate-limiting catalytic step in the trypsin-catalyzed hydrolysis of ZLNPE on changing the pH value from 2.5 to 7.8. At pH 2.5 the k + ~ step (deacylation) is rate-limiting in catalysis, whereas for pH values higher than 4.8, k+p (acylation) becomes rate-limiting.

Aiming to expand this initial observation, a detailed study

‘ The abbreviations used are: ZLNPE, a-benzyloxycarbonyl-L- lysine-p-nitrophenyl ester; ZANPE, a-benzyloxycarbonyl-L-alanine- p-nitrophenyl ester.

12449

12450 p H Effects in Trypsin Catalysis

of the pH dependence of the kinetic parameters of Scheme I was carried out with a cationic and a noncationic substrate and the results are presented here.

It will be shown that, with ZLNPE, the value of K, is pH- dependent below pH 5, implying a shift in pK of a group from 4.55 to 3.75 on binding of ZLNPE to trypsin. In the same pH region the value of k+z for this substrate depends on the ionization of a group with pK 3.75.

Such pH effects are absent in the trypsin-catalyzed hydrol- ysis of a-benzyloxycarbonyl-L-alanine-p-nitrophenyl ester, but in this case they reappear when ethylamine is present in the reaction.

Parallel experiments with a-chymotrypsin show that the rate-limiting step (acylation) for the catalyzed hydrolysis of ZLNPE and ZANPE is pH-independent between pH 3 and 6. In addition, no effect of ethylamine is observed in a-chymo- trypsin.

MATERIALS AND METHODS

Bovine &trypsin was obtained from Sigma. The concentration of trypsin was determined using the extinction coefficient at 280 nm E;?,,, = 15.4 (12). Bovine a-chymotrypsin was obtained from Worth- ington. The concentration of a-chymotrypsin was determined using the extinction coefficient a t 280 run E% = 20.2 (13). a-Benzyloxycarbonyl-L-lysine-p-nitrophenyl ester, a-benzyloxy-

carbonyl-L-alanine-p-nitrophenyl ester, a-benzyloxycarbonyl-L-ala- nine, and a-benzyloxycarbonyl-L-lysine were obtained from Sigma. Ethylamine and p-nitrophenol were from Merck. Ethylamides of a- benzyloxycarbonyl-L-alanine and a-benzyloxycarbonyl-L-lysine were synthesized according to Jones et al. (14). All reagents were of analytical grade and used without further purification.

The pH dependence of the spectral properties of ZANPE and of the product of the hydrolysis are superimposable with those of ZLNPE reported previously (1). The second order rate constant for the alkaline hydrolysis of ZANPE OH) has a value of 126 M" s" at 20.5 "C (E, = 24 kcal/mol, AS$ = -50 entropic units). For pH values below 7 alkaline hydrolysis of ZAN.PE is negligible for the time course of interest.

The following buffers were used phosphate (2-3.5), citrate (pH 2- 3.5), acetate (pH 2.5-6), and phosphate (pH 6-8.5), all at 0.1 M. pH values were measured with a Radiometer 51 pH-meter. The trypsin- catalyzed hydrolysis of ZLNPE and ZANPE was followed making use of the spectral changes associated with the formation of free p - nitrophenol. The optical density increase on hydrolysis of ZLNPE or ZANPE (350-400 nm) was monitored either in a double-beam spec- trophotometer (Cary 219) or in a Gibson-Durrum rapid-mixing stopped-flow spectrophotometer equipped with a 2-cm observation chamber.

Calcium was never presed in the reaction mixtures. Control ex- periments show that addition of calcium (chloride), up to M, did not modify the catalytic properiies of trypsin (15). Chromatography of substrates and products of trypsin and a-chymotrypsin-catalyzed hydrolysis of ZLNPE and ZANPE in the absence and presence of ethylamine (up to 0.2 M) were performed on precoated thin layer chromatography) plates of Silica Gel F254 (from E. Merck) using the following solvent systems (16): 1, butan-I-ol/acetic acid/water (155: 5 by volume); 2, ethyl acetate/pyridine/acetic acid/water (60.206:11 by volume); 3, chloroform/methanol/benzene (85105 by volume).

All the a-benzyloxycarbonyl-amino acid derivatives were located by spraying the chromatograms with a modified chlorine reagent (17). The a-benzyloxycarbonyl-L-alanine, a-benzyloxycarbonyl-L-lysine,p- nitrophenol, and the ethylamides of both a-benzyloxycarbonyl amino acids were used as markers.

The temperature was 21 k 1 "C throughout.

RESULTS

The Trypsin-catalyzed Hydrolysis of ZLNPE-The values of the kinetics parameters (within Scheme I) obtained from steady state and pre-steady state measurements (according to Refs. 18-20) are reported in Table I and, graphically, in Figs. 3-5.2 The data cover a range of pH from about 2.5 to 8.0.

The errors of k+2, K., kcat, and K,,, were evaluated according to the equation

The determination of the kinetic parameters of Scheme I is simplified by the fact that the process

k+l

k-1 E + S e E.S

may be regarded as being at equilibrium throughout the reaction, i.e k+z << k-, . Evidence for this is based on the observation that under the present experimental conditions no lag phase is observed in the rate of release ofp-nitrophenol from ZLNPE.

Fig. 1 shows the time course of the reaction of 60 PM trypsin with 4 p~ ZLNPE, i.e. [EO] >> [SO] (pH = 4.8, 0.1 M). Under these conditions the formation of E -S from E and S will be pseudo-fust order process characterized by a rate constant kloh given by Equation 1:

klob k + ~ . [ E o ] + k-I (1)

The absence of a lag phase means that the equilibration of E . S with E and S is complete within the 3-ms "dead-time'' of the stopped-flow apparatus so that klobs > 600 s-'. Given that K, (=k- l /k+ l ) has a value of the order 3.0 X M it follows that (i) k+l > 2 X lo6 M" s-l and (ii) k-' > 600 s-'. The prediction k-l >> kt2 is satisfied, since under the conditions of Fig. 1, ktP = 20 s-'.

A further simplifying condition arises from the observation that incorporation of low concentration (up to 40 p ~ ) of p - nitrophenol in the reaction mixtures did not affect the rate of release ofp-nitrophenol from ZLNPE. Hence, for Scheme I it is possible to write k-2 = 0. Similar arguments may be used also at the other pH values

to show that it is always possible to treat the formation of E - S from E and S as a rapid equilibrium process. When [EO] >> [So], with k-l >> k+2, the rate of p-nitrophenol release from ZLNPE should be a first order process with a pseudo-fist order rate constant:

The reaction is accurately Fist order for more than 95% of its course and the time course was independent of the wave- length in the range explored (350-400 nm). Furthermore, the value of klaPP was independent of substrate concentration as [Eo] > 5 X [so].

The values of k+z and Ks have been determined by Equation 2. Double reciprocal plots of l / k laPP against l / [ E o ] are shown in Fig. 2.

Fig. 3 reports the pH dependence of K+z values for the tryptic hydrolysis of ZLNPE. The best fit of data implies that two ionizing groups are involved in the pH dependence of k+:! with pK, values of 3.75 & 0.1 and 7.1 k 0.1, respectively.

The value of K, is pH-independent within the experimental errors in the pH range between 5.8 and 7.8. In the pH range between 2.55 and 5.8 the value of K, is pH-dependent, chang- ing from about 100 p~ at neutral pH to 800 PM below pH 3. The data may be fitted to a simple titration curve with a pK, value of 4.15 +. 0.1 (Fig. 4 ) .

Values of kCat/K,,, at different pH values obtained from steady state experiments are reported in Fig. 5 for comparison with values of k s s / K s determined from experiments where

incorporated into a standard Hewlett-Packard computer program used to evaluate the intercept and slope of plots of versus l / ( E o ] '(see Fig. 2), when [EO] >> [SO], and l/initial rate versus 1/[So], when [SO] >.> [EO].

pH Effects in Trypsin Catalysis 12451

TABLE I The values of steady state parameters and individual rate constant for the trypsin-catalyzed hydrolysis of ZLNPE

PH k,: K . k + J K . kS8 , K", k , . t lK , k+ t

S I B .%f s".p.w I s I BM S '.pM ' s ' 2.55 0.33 f 0.03 800 f 40 4.1 X 10 ' 0.05 f 0.005 250 f 30 2 x 1 0 ' 0.03 f 0.01 2.66" 0.395 795 4.9 x 0.033 0.015 3.01 4.0 f 0.5 780 f 30 0.005 0.30 f 0.05 200 f 50 0.0015 0.4 f 0.05 3.50 9.0 f 1 600 f 30 0.015 0.8 f 0.2 90 f 30 0.009 1 f 0.2 3.98 1 5 f 2 510 f 20 0.029 2.0 f 0.5 120 f 10 0.018 3.2 f 0.5 4.80 20 f 2 300 It 20 0.052 1 0 f 1 200 f 20 0.05 15 f 6 5.80 38 f 3 200 f 20 0.19 30 f 3 180 f 15 0.1; 200 f 100 6.80 83 f 8 93 rt 10 0.89 60 f 5 100 f 8 0.60 600 f 250 7.80 170 f 15 94 f 10 1.81 140 f 15 89 f 10 1.57 1600 f 400

From Reference 8.

T

J

J

- 1

0 100 200 300 rnsec FIG. 1. The time course ofp-nitrophenol release in the tryp-

sin-catalyzed hydrolysis of ZLNPE with [Eo] >> [So]. Trypsin was 60 VM and substrate 4 PM, acetate buffer pH = 4.8, I = 0.1 M at 21 f 1 "C, h = 370 nm. The arrow denotes the point where flow stops. The total absorbance change corresponds to 0.014.

[Eo] >> [So]. The data show a good fit to two ionizing groups with pK, values of 4.5 f 0.1 and 7.1 & 0.1, respectively. The correspondence of the values obtained by separate methods provides further evidence for the conformity of the system to the simple three-step model (Scheme I).

The values of k+:l were calculated by substitution of values k+2, K,, k,.,, and K,,, into Equations 3 and 4 (20):

The values of k+:l calculated from Equations 3 and 4 are the same within the errors.

Fig. 3 shows the pH-dependence of /z+:~. The data conform to a single ionizing group with pK,, value of 7.1 k 0.1.

Under the conditions where [SI)] >> [Eo], the trypsin-cata- lyzed hydrolysis of ZLNPE follows Michaelis-Menten kinetics. Steady state parameters (kc", and K,) have been determined from the intercepts on the ordinate and abscissa, respectively, of a plot of l/initial rate uersm l/[Sl,].

When [So] > [E0], the differential equations arising from Scheme I may be solved (20) to describe the time course of P I

O 5 r 1

FIG. 2. The variation of (klePP)" with [E,]" at different pH values under the condition [E0] >> [So]. The analysis is based on the reciprocal form of Equation 2 which predicts linear plots of slope

3.98 (A), 4.80 (O), and 5.80 (W). Acetate buffers 0.1 M; t 21 f 1 "c. K,/k+? and intercept on the ordinate Ilk.. The data are for pH values

release in the early stages of the reaction. The resulting expression is given in Equations 5-7:

where

and

Equations 5-7 predict, when k+? > k + , a "burst" of p - nitrophenol release of amplitude A (= a[EO]) with a first order rate constant k.

Fig. 6A shows that such a burst does occur at pH 4.8 when [So ] = 4 X [E,,]; the amplitude and the rate constant of the burst are A = O.lS.[S,,], i.e. 0.55.[RI], and k = 11.5 s", respectively. The predicted values from Table I and Equations 6 and 7 are in fair agreement, considering the variance of the kinetic parameters reported in Table I, and the uncertainty in the enzyme concentration (210%).

At pH values higher than 4.8 where k+e is rate-limiting in catalysis << the amplitude of the burst phase should be neglible (see Equation 6). The pH dependence of the burst amplitude reported in Fig. 6B shows that, as predicted, the

12452 p H Effects in Trypsin Catalysis

-2 t - 3 . I , , , 1 , . 1

2 3 4 5 6 7 8 9 1 0

PH FIG. 3. pH dependence of log k+, (0) and log k+s (0) for the

trypsin-catalyzed hydrolysis of ZLNPE. The data imply that two ionizing groups are involved in the pH dependence of k+z with pK, values of 3.75 f 0.1 and 7.1 f 0.1, respectively. The values of k+3 fit to a single ionizing group with pK, value of 7.1 f 0.1. Values of k+? (0) and k+S (m) obtained from Ref. 8 have been included for compar- ison. The data are for citrate and phosphate (pH 2-3.5), acetate (pH 3.5-6), and phosphate (6-8.5). All = 0.1 M; temperature, 21 f 1 "c.

2Ks 1 2 5

20 1 I 1 2 3 4 5 6 7 a

pH

FIG. 4. pH dependence of log Ks for the tryptic hydrolysis of ZLNPE (0) and ZANPE (A). Values of K, for ZANPE (7) and for ZLNPE (a), obtained from Ref. 21 and 8, respectively, have been included for comparison. Values of K, for trypsin-catalyzed hydrolysis of ZLNPE (0) and ZANPE (A) obtained from steady state parameters and k f Z according to K,$ = (&)/(kcat) .k+P. The solid line is a theoret- ical titration curve for a group of pK, = 4.15. For experimental conditions see Fig. 3.

, i , I 2 3 4 5 6 7 8 9 1 0

PH

FIG. 5. pH dependence of log k+,/K, for the trypsin-cata- lyzed hydrolysis of ZLNPE (0). Circles represent the data obtained from the experiments conducted with [EO] >> [SO]. Values of log kc,,/ K,, calculated from steady state measurements (0) have been in- cluded for comparison. A value of log k+n/Ks (0) has been obtained from Ref. 8 and reported for comparison. The solid line is a theoretical curve for two ionizing groups of pK, values of 4.55 and 7.1, respec- tively. For experimental conditions see Fig. 3.

B 1

PH FIG. 6. The burst of p-nitrophenol release during the tryp-

sin-catalyzed hydrolysis of ZLNPE with [SO] > [EO]. A , Trypsin was 60 pf and substrate 250 p ~ , acetate buffer pH = 4.8, 0.1 M, T = 21 * 1 "C, X = 370 nm. The inset shows the time course of the burst phase obtained from the overall progress curve by subtraction of the steady state phase. The amplitude of the burst is A = 0.16.[S0], i.e. 0.55. [EO]. The apparent f i t order rate constant for the burst phase is k = 11.5 s". B, pH dependence of the burst amplitude of p - nitrophenol release during the trypsin-catalyzed hydrolysis of ZLNPE (0). At all pH values trypsin was 60 p~ and ZLNPE 250 p ~ , h = 370 nm. Experimental value of burst phase (0) has been reported for comparison from Ref. 8. For experimental conditions see Fig. 3.

burst is virtually undetectable at pH values higher than 5.80. The middle point of transition corresponds to the pH value at which the change in rate-limiting step occurs. The Trypsin-catalyzed Hydrolysis of ZANPE-The de-

pendence of the initial velocity on the concentration of ZANPE follows simple Michaelis-Menten kinetics. Complete analysis of the time course of hydrolysis at a single initial substrate concentration yields values of kcat and K, in accord- ance with initial velocity estimates at different substrate con- centration. Over the wide range explored (2 X lo-' M-2 x M) the initid velocity is strictly linear with enzyme concentra- tion.

Between pH 3 and 8, the fit of data for kcat with a simple pH transition yields an average pK, of 6.90 rt 0.1 and kc,, (limiting) = 7.5 s-'. The values of K, are pH-independent within experimental errors and yield an average value of about 100 pM.

The kinetic parameters for the trypsin-catalyzed hydrolysis of ZANPE, obtained in the framework of Scheme I, are reported in Table I1 and Figs. 7 and 8.

The experimental data indicate that the formation of E.S complex is complete within the 3-ms "dead time" of the stopped-flow apparatus, following a pseudo-equilibrium proc- ess with a minimum value for the rate constant for the formation of E-S complex of 6 X lo6 M" s".

The value of K, has been found to be pH-independent throughout the wide pH range explored with an average value of 120 p~ (Fig. 4) . The observed value of K., is similar to that reported for trypsin-catalyzed hydrolysis of ZLNPE a t pH values higher than 5.8.

Fig. 7 shows the pH dependence of k+z and k+,3 for the trypsin-catalyzed hydrolysis of ZANPE. Data obtained from Kassera and Laidler (21) have been reported for comparison. The data indicate that only one ionizing group is involved in the pH dependence of k+z and k+3 yielding an average pK, value of 6.9 k 0.1. The observed pK, value is in fair agreement

pH Effects in Trypsin CataZysis 12453

TABLE 11

3.02 0.005 f 0.003 3.68 0.01 f 0.005 4.43 0.041 f 0.005 5.90 0.69 f 0.05 5.91 0.698" 6.08 1.12" 6.41 1.18" 6.62 2.30" 6.80 2.90 f 0.1 6.90 3.55" 7 10 4.88"

148 f 15 140 f 20 120 f 20 96 f 10 97.6"

3 X 0.005 f 0.003 130 * 20 3 X 10-~ 0.04 f 0.01

4.1 X 0.03 100 f 10 2.7 X 10-~ 0.2 f0.03 8 X 0.008 120 f 20 5.7 X 10-~ 0.06 It 0.01

0.0070 0.46 f 5 72 f 8 0.0065 4.2 f 1 0.0071 0.45 & 5 70 f 10 0.0065 4 f l

117" 0.0096 110" 0.016 107" 0.021

110 f 10 0.026 1.5 f 0.05 80 f 10 0.019 12 * 3 119" 0.029 148" 0.033

7.41 7.85 6.30 f 0.3 110 f 10 0.06 5.2 * 0.2 85 f 8 0.062

. ." 5.76" 100 f 10 0.058

25 i 5

From Reference 21.

PH FIG. 7. pH-Dependence of log k+z (0) and log k,, (0) for the

trypsin-catalyzed hydrolysis of ZANPE. Values of k+, (0) ob- tained from Ref. 21 have been reported for comparison. The data imply only one ionzing group with an average pK, value of 6.9 * 0.1. For experimental conditions see Fig. 3.

- 1 ~

- 2 -

- 3 -

-4 -

-51 I 3 4 5 6 7 8 9

PH FIG. 8. pH dependence of the log k+,/K, for the trypsin-cat-

alyzed hydrolysis of ZANPE (0). Open circles represent the data obtained from the experiments conducted with [Eo] >> IS,,]. Values of log kCat/Km calculated from steady state measurements (0) have been included for comparison. Values of log k+n/Ks obtained from Ref. 21 (0) have been included for comparison. The solid line is a theoretical curve for one ionizing group of pK, value of 6.9. For experimental conditions see Fig. 3.

with those reported for k,? in the tryptic hydrolysis of ZLNPE and N-transcinnamoyl-imidazole ( 2 2 ) .

Values of kCat /K, at different pH values are reported in Fig. 8 for comparison with values of k+n/K,. The data correspond to a simple titration curve with a pK, value of 6.9 I+_ 0.1.

The most significant result in trypsin-catalyzed hydrolysis of ZANPE is that k + ~ remains the rate-limiting step over the whole pH range explored (3-8), although its value changes with pH. The acylation ( K + J and deacylation (k+a) steps have

1

4a: 7 1 I o g K e

!,*M) 35 - D L ! - . . i

2 3 4 5 6 7 PH

FIG. 9. pH dependence of the dissociation affinity constant ( K , ) for the reaction of ethylamine with trypsin (0). Values of the inhibition constant, K, , of the ethylamine against the hydrolysis of ZLNPE have been included for comparison (0). Data of K , ob- tained from Ref. 24 (0) have been included for comparison. For experimental conditions see Fig. 3.

the same pH dependence and hence k+:j is always greater than kt2. Thus, the "burst" of p-nitrophenol release is undetectable at all pH values.

Effect of Ethylamine on the Trypsin-catalyzed Hydrolysis of ZANPE-It is known that the rate of the trypsin-catalyzed hydrolysis of "neutral" substrates is increased in the presence of alkylamino compounds (23, 24). Therefore, it appeared of interest to investigate the pH dependence of the hydrolysis of ZANPE on addition of ethylamine. The presence of saturating concentrations of ethylamine has been found to increase the initial rate constant for the trypsin-catalyzed hydrolysis of ZANPE by about one order of magnitude over a wide pH range (3-8). Fig. 9 shows the pH dependence of the dissocia- tion affinity constant (K,) for the reaction of ethylamine with trypsin. The values of Kc, were determined from the accelera- tion of the trypsin-catalyzed hydrolysis of ZANPE by ethyl- amine (24). The pH dependence of K , may be fitted with a simple pH transition with a pK value of 4.1 k 0.1. The inhibition constant, Kt, of the alkylammonium ion in the hydrolysis of ZLNPE can also be considered as the dissocia- tion constant of the trypsin-alkylammonium complex. Very good agreement is found between values of K , and K , (see Fig. 9). The value of K,,, (-90 PM) for the trypsin-catalyzed hy- drolysis of ZANPE is independent of the absence or presence of ethylamine (up to 0.2 M ) between pH 3 to pH 8. This fact implies that the binding of the latter does not affect the binding of ZANPE (see Tables I1 and 111). Therefore the accelerating effect of the ethylamine is exclusively due to an enhanced activity of the catalytic site as reflected by the increase of kc,at.

The pre-steady state and steady state parameters for the trypsin-catalyzed hydrolysis of ZANPE in the presence of saturating concentrations of ethylamine are reported in Tables 111 and IV. The minimum value for the rate constant for the

12454 pH Effects in Trypsin Catalysis

TABLE I11 The values of steady state parameters and individual rate constant for the trypsin-catalyzed hydrolysis of ZANPE at saturating

concentration of ethylamine

PH k+r K , k+P/K* kcat K , k d K m k+a Ethyla- mme

S-1 PM S".pM" S" PM s " . p M - ' S" M

3.15 0.1 k 0.02 150 k 30 6.7 X IO-* 0.03 k 0.005 50 k 10 6 X 10" 0.03 f 0.01 0.2 6.5 6.7 f 1 120 f 15 0.055 4.5 & 0.5 80 k 10 0.056 14.5 k 5 0.04

TABLE IV The values of amplitude (A = a.(So/) and the time course (k) of

burst phase accompanying p-nitrophenol release during the trypsin-catalyzed hydrolysis of ZANPE at saturating

concentration of ethylamine pH E" SO A k

P M PM S"

3.15 60 250 0.12 0.12 6.5 60 250

formation of E . S complex in the presence of ethylamine is 4 X lo6 M" sec". The most significant result shown in Tables I11 and IV is the pH dependence of k+? and / z+~ which implies that at pH 3.0 k+a is rate-limiting in catalysis, whereas at pH 6.5, kt* becomes rate-limiting. Under the conditions of a large molar excess of substrate over the enzyme the burst of p- nitrophenol release is experimentally detectable at pH 3.0, where k+S is rate-limiting in catalysis (Table 111). At pH 6.5 where k,, is rate-limiting in catalysis the burst phase is absent. These experiments c o n f m the change in the rate-limiting catalytic step in going from pH 3.0 to 6.5 for the trypsin catalyzed hydrolysis of ZANPE in the presence of ethylamine.

Under all the experimental conditions the products of the enzymatic hydrolysis of ZANPE and ZLNPE, in the absence and presence of ethylamine (up to 0.2 M), are the correspond- ing a-benzyloxycarbonyl amino acids and p-nitrophenol. The ethylamide derivative of both a-benzyloxycarbonyl amino acids has never been observed when the analysis was done during the progress of the hydrolysis process.

At very low pH (-2.0) trypsin is inactive in the hydrolysis of ZLNPE and ZANPE. This effect cannot be accounted for by irreversible acid denaturation since the enzyme brought back to neutral pH shows complete recovery of the activity.

The a-Chymotrypsin-catalyzed Hydrolysis ZLNPE and ZANPE-The experimental observations indicate that the formation of a-chymotrypsin-substrate (E .S) complex is com- plete within the 3-ms "dead time" of the stopped-flow appa- ratus. The minimum value for the rate constant for the formation of E.S complex is 6 X lo6 M" s-' for ZANPE and 1 X lo6 M" s" for ZLNPE.

Steady state and pre-steady state parameters for a-chymo- trypsin-catalyzed hydrolysis of ZANPE and ZLNPE have been evaluated as previously reported for the trypsin-cata- lyzed hydrolysis.

The kinetic parameters (see Scheme I) for the a-chymo- trypsin-catalyzed hydrolysis of ZANPE and ZLNPE are re-

ported in Table v. In this respect the most significant results are (i) the pH independence of the values of K, for both the substrates, between pH 4.0 and 6.0, showing average values of 120 pM and 700 PM, respectively; (ii) the pH independence in the rate-limiting step ( k , ~ ) over this pH range in agreement with observations on the a-chymotrypsin-catalyzed hydrolysis of anilides (25); thus, the burst of p-nitrophenol release is undetectable at all pH values; and (iii) the absence of any effects of ethylamine on the rate-limiting catalytic step con- trary to what was reported in the case of trypsin.

DISCUSSION

The pre-steady state results indicate that the kinetic data for the trypsin-catalyzed hydrolysis of ZLNPE may be con- sistently fitted to the simple three-step mechanism (Scheme I) over a wide pH range between 2.5 to 8.0. However, it should be emphasized that Scheme I is only minimal and other more complex models may apply as long as certain conditions are satisfied

According to Peller and Alberty (26) the pH dependence of the ratio k+,/K, (= kCat/K,) should reflect ionization of group(s) in the free enzyme. The pH dependence of the ratio k+*/K, depends on two ionizable groups with pK, values of 4.5 f 0.1 and 7.1 k 0.1, respectively. The inspection of the amino acid sequence (27) and of the three-dimensional struc- ture of the active site of trypsin (28-30) suggests the assign- ment of the observed ionization equilibria to Asp-177 and His- 46. Asp-90 involved in the active site of trypsin is not likely to be responsible for the observed acid-base equilibrium in the pH range between 2.5 and 5.8. In fact the protonation of Asp- 90 may be expected to be associated with the inactivation of the enzyme (31-33).

It is noteworthy that values of pK, determined from the pH dependence of the ratio kcat /K, are in fair agreement with the corresponding values obtained from the pH dependence of the ratio k+s/Ks from the experiments with [EO] >> [SO].

The k+2 versus pH profile implicates two ionizing groups with pK, = 3.75 f 0.1 and 7.1 f 0.1, respectively, in the E-S complex.

The results reported here imply that there is a pK shift of an ionizing group in the enzyme in the acid pH region on binding of the substrate. This should be reflected in a change of K, with pH in the acid pH region. According to linkage relations (34) the change should be about 5-fold, going from pH 5.8 to 2.55. This expectation is in good agreement with the observed increase of the value of the dissociation equilibrium

TABLE V The ualues of steady state andpre-steady state parameters for the a-chymotrypsin catalyzed hydrolysis of ZLNPE and ZANPE

pH kt1 K , k + d K , , kc,, K", kvat/Km k+:x Substrate S -' P M s".pM" S" P M S " . P M - ' 8-1

4.00 0.06 f 0.01 700 f 50 8.5 X 0.04 f 0.01 640 f 40 6 X IOw5 0.3 -t 0.1 ZLNPE 5.95 3.8 f 0.5 700 k 50 5.4 x lo-" 2.8 f 0.3 620 f 40 4.5 x lo-' 18.2 & 6 ZLNPE 4.05 0.004 c 0.001 I20 f 10 3.3 x lo-' 0.003 f 0.001 100 -c 10 3 x 0.016 f 0.003 ZANPE

4.00 0.005 f 0.001 115 f 10 4.3 x 0.003 f 0.001 105 f 10 3 x 0.018 -t 0.003 ZANPE (+ethyls-

6.05 0.5 k 0.1 120 f 10 4.2 x 10"' 0.25 f 0.05 85 f 10 3 X lo-' 1.5 -t 0.3 ZANPE (+ethyls-

6.00 0.50 f 0.1 125 f 10 4.0 X IO-'' 0.3 f 0.05 90 f 10 3.3 X lo-' 1.0 f 0.3 ZANPE

mine 0.2 M )

mine 0.2 M )

pH Effects in Trypsin Catalysis 12455

constant (K,) going from pH 5.8 (Ks = 200 f 20 PM) to pH 2.55 (& = 800 40 p ~ ) . On the other hand, the neutral ionization (pK, = 7.1 -+ 0.1) does not undergo a shift in the formation of the enzyme-substrate complex. Accordingly, the value of K, is pH-independent within experimental errors in the pH range between 5.8 and 8.0.

The change in pK of the acid group (most likely the carboxyl group of Asp-177) on binding ZLNPE probably results from an electrostatic interaction with the €-amino group of ZLNPE. This conclusion is in agreement with (i) the pH independence of Ks for the hydrolysis of ZANPE and (ii) the pH dependence of the dissociation constant of the trypsin-ethylamine com- plex. The latter finding implies a pK shift, on binding of ethylamine, similar to that observed with ZLNPE.

Similar behavior has been previously reported for the equi- libria of bovine trypsin with benzamidine (35).

The relative increase in the value of k+z with respect to k + ~ in the catalyzed hydrolysis of ZLNPE at acid pH, and the increase in k+z for the hydrolysis of ZANPE in the presence of ethylamine, indicate that the ionized form of Asp-177 plays a role in the acylation but not in the deacylation step.

To obtain independent information about the role of Asp- 177 in tryptic catalysis, steady state and pre-steady state parameters for the a-chymotrypsin-catalyzed hydrolysis of ZLNPE and ZANPE have been measured between pH 4.0 and 6.0. The only significant difference between the catalytic sites of the two proteases is that the neutral serine 189 in a- chymotrypsin (36-38) has become the negatively charged aspartic acid 177 in trypsin. The absence of Asp-177 at the active site of a-chymotrypsin is in agreement with the pH independence of both the K, and the rate-limiting step (acyl- ation) for ZANPE and ZLNPE in the acid pH range and with the absence of effects of ethylamine.

The results reported here allow three significant conclusions related to the catalytic mechanism of trypsin. (i) The affinity of cationic ligands for the active site depends on the state of ionization of an acid group of the protein. According to linkage relations the change of affinity with pH reflects a pK shift of the acid group from 4.5 f 0.1, in the free enzyme, to 3.75 f 0.1 in the trypsin-cations complexes. This linked group is, most probably, the carboxylic group at Asp-177. (ii) The ionization of the linked acid group affects the rate of acylation of the substrate. (iii) The large differences in the pH dependences of the trypsin- and a-chymotrypsin-catalyzed hydrolysis of ZANPE and ZLNPE at low pH may be related to the struc- tural differences at the active site of the two proteases, in particular to the existence of the carboxylic group at the position 177 in trypsin.

Acknoudedgments-We thank Dr. M. Bolognesi who read the manuscript and offered constructive criticism, and Dr. E. Menegatti for the chromatographic analysis.

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