The effect of the N-acyl moiety of the substrate on α-chymotrypsin binding and catalysis

JortrnaloFh#oLecuIor&ialys&. 6 (1979) 235 - 249 @ Ekevïer Sequoia S.A., Lausanne - Rinted in the Netherlands

THE EFFECX OF THE N-ACYL MOIETY OF THE a_CHYMOTRYPSlN BINDING AND CATALYSIS

R. J. COLL=

SUE?STRATE

ON

and J. R. WHïTAKER**

Depar:merrto/Food 95616 (U.S.A.) (Receiwd

235

Science

and

Tecknology,

University

o/Califomïa,

David. California

December 12.1978)

Summary The effect of the N-acyl moiety of the substrate cn a-chymotrypsin binding and catalysis of N-carbomethoxy-, N-formyl-, N-acetyl-, N-chloroacetyl-, N-methancsulfonyland unacylatcd L-tyrosine ethyl esters was studied. Fresteadystate and steady-stati kinetics of the a£hymotrypsincatalyzed hydrolysis of L-tyrosine ethyl ester indicated that deacylation is the rate-limiting step at pH 7. However, at pH 5 the rates of acylation and deacylation were approximateïy equal. The rates of deacylation of the various acyl-chymotrypsins, forrned as intermediates with the ahove substrates, correlated with the hydrogen-bonding ability of the N-acyl substituent. However, the mtes of acylation of crchymotrypsin by these substrates did nat correlate with the hydrogen-bonding ability of the N-acyl substituent but showed some correlation with the abilih of the substrate to undergo azlactone formation. The dissociation constants of the various substrates with the enzyme do not correlate wïth the hydrophobicity, molar refractiv-ities or PK, of the parent acid of the N-acyl group, indicating that other portions of the substrate play a major role in binding also.

Introduction The mechanism of e-chymotrypsin catalysis bas been studied in more detail than that of any other enzyme. Gne particularly powerful technique has been the comparison of relative rates for a series of substra& systematically varied in one portion of the molecule. Figure 1 shows postulated points of interaction betneen the enzyme and substrate important in the formation *Present address: I11inois 60201. USA.

Department

of Chemistry,

h’orthwestem

UniVersitY,

Ew.IIs~~,

R. J. C. receivedfinanäal support through a schoInrshipfmm the Campbell Soup Campany during fhis research. *fContacE for reprints.

236

Fig. 1. Schematic representation of the activa site 01 Q-chymotrypsin. A. Michaelis complex bekveen enzyme and N-acyl tyrosine ethyl ester. 3. Postulated totrahedral intermediate. The atoms in bold lettering belang to the enzyme.

of the Michaelis complex and the transition state. Results obtained from such studies have been valuable in partially elucidating the nature of the transition states of the acylation and deacylation steps of whymotrypsin catalysis. Most studies of thïs type have involved comparïson of a series of substrates which vary in the les-ving group (R’ in Fig. 1). With ester substrates this techniquc bas provided evïdence for the acyl-enzyme intermediate [l] and bas helped to characterize the nature of an intermediate fonned prior to the acyl-enzyme, uti., the tetrahedral intermediate 12 - 4]_ The nature of the tetrahedral intermediate bas been studied in detail for anïhde substrates 15-71. For enzymes such as achymotrypsm, whose natural substrates are proteins, it bas been shown that enzyme-substrate interactions distal to the scissile bond (R group in Fig. 1) are involved in stabilizing the transition states of the reaction. Additlon of an amino acid to the N-terminus of phenylalanine amide can enhance the rate of acylation by three-fold [SI. However, there have been relatively few reported investigations on the mechanism by which the N-acyl moiety of the subs&& assists in cataIy.sïs. Steady state data from Û series of N-substituted L-tyrosine zmides indiczted for amide a linear relatronship between k,,, (equal to the rate of acylation substrak) ancï the mok refractivky of the N-acyl group [9] _ Thïs could be interpreted as evidente that London interactions are important at the enzyme-substrate lotus. Sawyer and Kirsch [ 101 studied the kïnetic isotope effect on hydroxide and Whymotrypsin
237

180/1aOethyl esters. While the isotope effects on the rates of of these two compounds were Identical, significant differences were found for the enzymatic reaction The results were interpreted as refkctig either different bond orders for the leavïng group oxygen in the two transition states or for different mechanisms of enzyme acylation by the two substrates_ The latter interpretation is based on evidente that Q-N-aCety1 esters with goed leavïng groups can undergo oxazohnone (tiactone) formation during hydrolysis whereas the correspondmg c+-N£arbomethoxy compo*unds cannot. Additional cvïdence that an azlactone inteermediatemay beformedin L+£bymo~psincatalyzdrractionsisgivenby the report of Coletti-Breviero ef 41. [ll] . They quenched the a-chymotrypsincatalyzed hydrolysis of N-furylacryloyl-L-tryptophan methyl ester and isolated a reaction intermediate identical, chromatographically, to the azlactone of N-furylac~~loyl-tryptophan. However, de Jersey et af. [lZ] were unable spectrally to observe an azlactone irrtermediate in the deacylation of hippuryl-a~hymotrypsin. Steady-state studies compting the rates of hydrolysis of a-acetamido, a-acetyl, and other aLsubstitut.ed L-ester substrates indlcate that the hydrogenbonding abïlity of this portion of the molecule is of prïmary importante in deacylatron [13,14] _ These observations support the postulacion, based on X-ray cryswographic studies 115 - 171, that a hydrogen bond is formed between the a-arnido hydrogen of the substrate and the carbonyl oxygen of serïne-214 on the enzyme (Fig. 1B). The present investigation was prompteed by the partial state of knowledge on the magnitude of the stabilizing influence of the a_N-acyl group on the transltion states of the e-chymotrypsin~atiyzed reaction end the possibïlity that the carbonyl group of the N-acyl grouping may act as a nucleophile toward the carbonyl carbon of the subsfxate (azlactxme formation). Formation of this intermediate might be assisted by hydrogen bonding of the amido hydrogen to Sr-214 of the enzyme [15 - 171. Comparison of the relative rates of acylation and deacylatïon for several N-acyl-L-tyrosïne ethyl esters varyïng in mok refractivïty, hydragen-bonding abïlity, hydrophobicity, and ability to undergo azlactone formation should assist in understanding the nature of the stabïlizing effect of the N-acyl moiety during catalysis. L-tryptophan

alkaiine hydrolyses

Experimental Ma termis c&hymotrypsïn (3X crystallized; CD1 345895) was from Worthïngton Biochemical Corp. Proflavïn sulfate (Mann Research Lab, T2828) was recrystallized twice from water and was protected from light durÏng storage. N-Methylchymotrypsin was prepared by Ryan and Feeney’s method 1181 except that the derivatives were put on the affinity column at pH 5 and eluted at pH 2.85.

Most of the N-acyl-L-Lyrosine ethyl esters were synthesized by the Schotten-Baumann procedure_ L-Tyrosine ethyl ester was dissolved in ethyl acetate and the solution was stured above an aqueous sodium bicarbonate solution. One equivalent of the acylating agent was added dropwise over a period of approximately 30 minutes. After three hours of reaction the phases were separated, the organrc phase was washed with dilute HCl followed by dilute sodium bicarbonate, and (after drying over anhydrous NazSO,) the solvent was removed ik uucuc. The initial crude ?J-methanesuifonyl (N-mesyl) product was coated wifh a yellow oïl which was removed by washing with severai smal1 portions of ice-cold chloroform. The h--formyl substrate was synthesized according to the method of Bergel and Lewis [lg]. AR the substrates werz pure by thin layer chromatography on Silica Gel G plates developed with n-butanol:acetrc acid:water (4:1:1, v/v) and benzene: methanol (75:25. v/v). The compounds were detected by iodme vapor. IJ-Acetyl-L-tyrosine ethyl ester melted at 89 - 94 “C (reported m.p. 84 - 87 ‘C [ 201). N-Carbomethosy-L-tyrosine ethyl ester, recrystallized twice from diethyl ether-petroleum ether, melted at 87.5 - 89 “C (no prevïously reported synthesis). N-Chloroacetyl-L-tyrosine ethyl ester, recrystallized from dichloromethanerpetroleum ether, melted at 79 - 83 “C (reported m.p. 86 - 87 “C [ 21])_ N-Formyl-L-tyrosine ethyl ester, recrystallized from chloroform:carbon tetrachloride, meltcd at 106-5 - 108 “C (reported m.p. 105 - 106 “C [lg] )_ N-Methanesulfonyl-L-tyrosïne ethyl ester, recrystallized from ethanolrpetroleum ether, melted at 153 - 156 “C (no previously reported synthesïs). Me thods Al1 the experiments, escept where noted otherwïse, were performed at 26 5 OC, pII 5.0, and J.I= 0.2M. Dlfference spectra and binding studies were performed on a thermostatted Cary 118C recording spectrophotometer. Steady-state experïments were performed on a thermostatted pH-staat, consisting of a Radiometer pH-25 pH meter, titrator 11, and microelectrodes (G2222C and K4112), under barium hydroxide-scrubbed nitrogen. The ionic strength was adjusted to 0.2M with NaCl The stock enzyme solution was prepared darly m 1mM HCl and was stored on ice_ The actlve enzyme concentration was determined by spectrophotometric titration with 2-hydroxy5-nitro-e-toluenesulfonic acid sultone [ 221. The reaction was initiated by the addition of 25 ~1 of stock enzyme solution to 5 ml of temperatureequrhbrated substrate solution- Tnitial rates of hydrolysis were measured, usually in trïplicnte, for each substrate concentration by determining the rate of addition of standardized O.Oi or 0.051M NaOH requïred to mamtaïn the pH constant at 5.0. NO vessel wal1 effects were observed [23]. Pre-steady-stati experïments were perEormed using a thermostatted Gibson-Durrum stoppcd-flow spectrophotometer with a deadtime of 5 ms as dctermined

by the method

of Gutfreund

[24J.

Freshly

prepared

substrate

and buffer solutions were degassed in uucuo prior to use. Except where noted othenvise, al1 stopped-flow experïments were performed with 0.l.M

239

sodium acetate buffer, pH 5.0, adjusted to 0.2M

ionic strength wïth sodium

chloride. For the nroflavin dïsplacement studies, enzyme was dissolved in proflavïn-buffer solution. The fInal concentrations (during the reaction) of enzyme and proflavin v-‘ere, for most experiments, 20pM and 50 or 58pM, respectively. The reactio,x components were always equilibrated to constant temperature prior to mixing. fn each experiment three replicates were perfonned at each substrate concentratïon. The reported values for kz and K, are averages of at least three separate experimentc. For stopped-flow

expenments with unacylated substrates at pH 5-0, the celi was flushed wïth buffer immediately after the reaction to prevent precipitation of the poorly soluble product, tyrosine. The hydrophobicity constants, ik, were determïned by the mcthod of Hansch [25] _ 2mM solutions of the substrates, prepared in pH 5.0 acetate buffer, P = 0.2M, were extracted with an equal volume of n-octanol. The corlcentrations of substrAti in the two phases were determined according to Fujita et al. f26] _ N-Acetyl-L-tyrosïne ethyl ester was used as the reference compound for the cakulation of X. of resdts rates obtained from the steady-state experïments were plotted accordmg to the unweighted Eadie-Hofstee method using a Hewlett-Packard statistical program and plotter. Final values for k,,, and IC, were determined using a taped program of the bilinear regression analy& of Wilkïnson 1271 with a Wang 700 computer. The kcat values for L-tyrosine ethyi and methyl esters obtaïned at pH 7.0 were corrected for proton uptake by the e-amino group of the product by the method of Purdie and Benoiton [28] since the pK, of the a-amino group changes from -7 in the substrate to -9 in the product. The kïnetic equations used for the ana&is of data from stopped-flow proflavin dispiacement expemments weArethese derived previously [29]_ Fir,t Qrder trans;ent.s of profla-ti absorbsnce at 465 nm were retieved from the Tektronix storage oscilloscope by photographing wïth 35 mm High Contrast Copy film. The negative was then placed in a photogrzphic enlarger and the signal was traced onto graph paper for calculation of the fïrst order ratte constant, boiS_ Initially, S2 and K, were calculat-ed by plotting kobs k czt US- (kobs - kcpL)/Cs according to the unweighted method of EadieHofstee. Fcr these substrates soluble to concentrations suffrcrently greater than theïr K, values the Wilkïnson bilinear regression analysis was then used. For all substrates studied the kobs values determined at low concentratïons of substrate deviated from the straight line relationshïp obtained at higher at which this nonhnearity values of kobs_ The subskrate concentrations

Calculatinn Initml

occurred proflavin.

were not influenccd by changïng the concentration of enzyme or Only data for substrate concentntïons above this devïation were

used to calcrrlate the kïnetic constanr_s. The N-methanesulfonyl substrate was not solubk at concentrations above the nonlïnear regïon. Therefore, the value of Ks for thïs substrate was

240

determïned by proflavïn dïsplacement from N-methylchymotrypsin, a method which has been shown tc give K, values identical wïth these obtained by the abave procedure [30] _ The value of k, for the N-methanesulfonyl substrate was then calculaAt. from the relation k,/K, = k,,JR,. K, for Ltyrosine ethyl ester at pH 5.0 was also determmed by proflavin displacement from N-methyl£hymotrypsïn. For these substrates for which ka + k cat, the k, values were calculated from the determined values of kcat and k, using the relation, k;i, = ka1 + kil (kcaL = k2k3/(kp f ka)).

Results The results wil1 be presented and discussed according to the generally reactions represented in acceptecl mechamsm of a-chymotrypsm~atalyzecl eqn- (1)

R,

E+S,-‘E-Sd

k2

E-Acyl

s

E + P2

(1)

2 +

PI

E- S is the noncovalent Michaelis complex; E-Acyl is the acyl-enzyme; P, is ethanol for the substrates used in this study, and PL is N-acyl-Gtyrosine. The steady-state rate constant, kcac, has the reïation to k, and k3 of ki& = ksl + kzl and the Michaelïs constant, K,, 1s equal to K,(k3/(k, + ka))where

Steody-state

cxperiments;

determination

of K,,

kcmt and k3

Steady-state data for Ltyrosme ethyl and methyl esters at different pH values are gïven in Table 1. Ako shown for comparison in Table 1 are data for Ltyrosine p-mtrophenyl ester [31]_ K, was determined to be 0.6 K, (the laar measured by stopped flow spectiophotimetry and by displacement of proflavin from N-methyl£hymotrypsin) at pH 5.0, while kcaL was determined to be 16 s-l. From the relations of K,, IC, kcat, k2 and k3 (eqn. ti)), k2 and k3 were calculated to be 32 s-l_ Lïsted in Table 2 are the steady-state rate data at pH 5.0 and 26.5 OC for the substrates studied, a.long with values for the hydrophobicity, molar refractivïty and the p& of the parent acid for the different N-acyl substituents. Also included in Table 2 are the values for k,, the deacylation rate constant. Figure 2 shows a plot of log k, U.S.the pR, of the parent acid of the N-acyl moiet-, ; the R2 vzlue IS 0.76 (R = correlation coeffCent)_ Molday and Kallen 1341 and Sheïnblatt and Rahamïn [35] have shown that the log of the rate of hydrogen exchange varGzs lïnearly with the p& of the parent acid for a series of znides. Therefore, in our analyses we assumed that the hydrogen-bonding abihty of the N-acyl moiety varies as the p.&, of the parent acid. Ingles and Knowles [ 133 observed that the deacylation rate using ìVacetyl-Lphenylalanïne ethyl ester was much faster than that for its onygen counterpart, O-acetyl+-phenyl-Glactic acid ethyl ester, yet one would expect

241 TABLE

1

Steadyrrtate fineti= for a-chymotrypsincatalyzed and p-nitrophenyl esters Ester

Substrate renge (mM)

Methyl Ethyl

10.0

Methyl Ethyl p-Nitrophenyl* MethyILf Ethyl** p-Nitrophenyl*

100-

- 100

100

PH 4.5 4.5

6.3 I 0.2 5.7 = 0.5

2.5 - iO0 2.5 -100

5.0 5.0 5.02

15.4 = 1.4 16.3~ 0.5 91s f 3.0

1.0 -16 1.0 - 12

7.0 70 6.98

45.8 r 1.7 38.2 * 1.5 35.0 c 10

_

_

hydr~lysk of L-tyroeine ethyl, methyl

30.8 = 3-3 43.5 5 10.0 22.0 * 5.0 36.5 f 2.8 2.67 5 0.28 3.32 5 0.36

*FromKézdyetal. [31]. was corrwted according to Purdie and Benoiton 1281 for the differente In pK, of the earruno moup of the substrate znd product.

**kat

the opposite

from electron withdrawal considerations. Therezore, there not appear to be a free ene= relationship with the electron kthdrawal the Wacyl goup.

does of

flow experiments; determimtion of K, arrd k, The method used for monitorïng the acylation step was the proflavin dispkacement technique [29] _ PrGflavin competes wïth substrate for binding to the enzyme. Enzyme-substrate intermediafes, includïng the noncovdent _Michaelis complex and the covalent azyi-cnzyme, do not bind proflavin Roflavïn bound to the enzyme bas a higher absorbancz at 465 nm than does free proflavin, thus provïding a special means of monitorïng acyl-enzyme production. The dissociation constant of proflavin and enzyme was de& mined by steady-state competitive inhibition studies to be 109yM, in good agreement with other values dete_rmined at pH 5.0 (99pM [36 ],112~M 131). Upon mixïng substrate w-ith enzyme and proflavin in the stopped-flow apparatus, we observed an immediate decrease in absorbance at 465 nm, completed wiihïn the deadtime of the apparatus, due to Michaelis complex formation. The immediate jump ís followed by a fïrst order decrease in absorbance to a steadystate level. Thïs transient reflects the btid up of a population of acyl-enzyme [29; our own observations] _ The magnitude of the immediate decrease in & as is a function of the substrate concentration relative to K, (our observations). The amplitude of the first order transient reflects the ratio of k2 ti ka_ Table 3 lists the amplitudes of the Lirst order kansïents (in log(voks)) for companson among the substrates. These amplitudes were deterrrmined under the conditions of 2OpM enzyrne, 58~M proflavin, and substrate concentxation equal to IC, (app) [= K, (1 + R~flavirr/I(~,~~~~~~)~, so that

Stopped

242

-~ o

=

~ E]

,.-3 --

cs

I

[]

r-~

z.O

,~.¢~. ~-.

.

f--

o

.o~.~

eml i:,,-)

N

N

-i-i

-t-i

+i

e4

~-i -

O

E

~

~

N

2 ,

,

.

O

,

.

.

,

_

I.~_

,

L~

~,_T,

.~

~.~

243

0’ 0’

4

2

6

I

6

Flg_2. Plot OF log k, US. the pK,

OF the parent

scid of the

N-2131

moiety- N-Mesyl is

N-methanesulhrnyl.

half the total enzyme would he present either as the noncovalent Mïchaeiis complex or as the acyl-enzyme. The amplitudes of these transients are useful, quaKatively, in mterpreting some of the results. K, and .Ezsvalues determined by stopped-flow proflavin dïsplacement experïments for the N-acyl-Ltyrosme ethyl ester substrates are also @ven in Table 3 along wïth kcat and IC,,.,(app) values of the correspondmg amide substrates, as determïned by steady state techniques [37] _ For amide substrates k3 > k, so K, (app) is approximately equivalent to IC,_ The k2 for the N-methanesulfonyl substrate was determïned by equating .%a/KS to kcat/Km and was not directly observed because of limited substrate solubfity. The value of k, calculated in this marmer is supported by comparison of the transient amphtude with those for other substrates. As shown in Table 3( the transient amplitude for the N-methanesulfonyl substrate (0.25 log(volts); calctited kz/ks = 4.5) is between that of the N-formyl substrak (9.22 log(volts); kz/ka = 3.3) and that of the N-carbomethoxy substrate (0.30 log(volts); kz/ka = 12.7)_ The determined acylation rate constants for the series of N-acyl-Ltyrosine ethyl esters used in the present investigation are compared in Table 4 with the rates of dkahe hydrolysis for a series of N-acyl glycine p-nitrophenyl esters as determïned by de Jersey et uZ_ Cl.21. de Jersey et UL reported thai al1 the N-acyl glycine p-nïtrophenyl esters, with the exception of the Ncarbobenzoxy and N-tosyl substrams, hydrolyzed uia azlactone formatïon. It is expected the Ncarhomethoxy compound used in onr investigation would behave stiarly to the AQarbobenzoxy compound in regard to azlactone formation. Under the alkaline conditiona of de Jersey et al. 1121 +he amido group of the N-tosyl glycine p-nitrophenyl ester would be almoti completeely ionized, as the amido p& is approximately 10 [39]. The value of kOH Tor the un-ionized tosyl compound is unknown; however, it should

244 TABLE

3

Re-steady Ltyroaine N-Acyl

state and steady state data for a-chymotrypsin~talyzd ethyl esters and amides

g-roup

Ethyl

hydmlysis

Anüds

esse&

K,

k2

(mM)

Cl)

N-ChIoroacetyl N-Acetyl N-Carbomethoxy N-Methanesulfonyl

13.3 2 2.7 10 i 1 6.8 ? 1.8 1 lc

170 69 18 31f

33 21 12.7 4.5

N-Fonnyl Unacylated

8? 60C

16 32’

3.3 1

1

of N-acyl-

kzlb3

Traruaent arnplitudeb log(volts)

G(~PPF

Llld

(ti)

s-1

0 40 0.35 0.30 0.25

27 32 6.5b

4.0 2.6 0.85b _

c.22 0

12 -

0.45 -

_

pFrom the present work. Reactions were at pH 5.0, 26.5 “C and ionic Aren& of 0 2M. bDetermined at substrateconcentrationequal to K,(app) =K,(l + Rof!!tin/Kp,Or~avin)_ =K,.,.,(epp) for the Nacyl-L-tyrosïnamides [37 ] as recalculated Erom data of Hein and sg protem nitmgen-r ml-’ 138 ] _ The measurements were made at pI-X values betwen 7.6 and 8.0. eDetenmined from proflavm displacement studies wnith N-methylchymotrypsin. = kzfKs as d-ibed in the teti_ fCalculated from tbe relationsbip kcat/K,,, values of K, = ‘Calculation based on rhe essumption that k 2 = ka and from detenrüned that k;it = kzl + ksl _ 0.6 K,, keaL = 16 s-l and the relationship hFor Nclarboethoxy-Ctyrosïnamide.

definitely be higher than for the ionized substrate. The N-methanesulfonyl substrate should behave similarly to the N-tosy! compound with regard to azlactone formation _ Bodanszky and Ondetti [ 401 indicated that electron withdrawing groups on the N-acyl substituent should enhance azlactone formation. Therefore, of the compounds studied, the N-chloroacetyl substrate wouId be expected to have the highest propensity toward azlactone formation. The determined k2 values for the o+chymotrypsir.-catalyzed reactions vary in the szme direction

as the propensity

for azlactone

formation

(Table

4).

DiscusGon Rate-determiningstep tyrosine esters

for

crchymotrypsincatulyzed

hydrolysisof

unacylated

Our stopped-flow spectrophotometric studies w-ith Gtyrosine ethyl ester at different pH’s indicated that there may be a change in rat+limiting step with pH for this substrate. At pH 5, 50~M enzyme, 50~M proflavin and 50mM Gtyrosine ethyl ester (Km = 36.5mM) no transient was observed, indicating that k, s kJ. Under the same conditions, the size of the dient increased w-ith increase in pH (transient amplitudes, logged by the instrument, were 0.160 and 0.360 volts at pH 5.5 and 7.0, respectively) indicating that

345 TABLE

4

Compsison OF acylatios rat9 of ct-chymotrypsin group t.o undergo azlactone Formation N-Acyl

ty-rusine ethyl

N-Acryl

groep

abfity

ester*

N-Acyl

glycine-p-nitrophenyl

k,

N-Acyl

group

(s-l 1

Chloro~cetyl Acetyl Formyl Carbomethoxy Methanesulfonyl Unacylated

tith

170 69 16 15 31 32

Benzoyl Cin-oyl Acetyl Formyl Czrbohenwxy Tosyl

of N-acyl

ester**

Jzo-ti*+* (M-1 s-l) 7 040 3 480 1110 480 156 38

*Dctcrmined in the present nrork. **Data from de Jersey et al [12]_ ***Rats of p-nitrophenol release. Al1 the compoundz except the Ncsrbobenwxy and N-tosyl glycine p-mtrophenyl esters hydrolyzed an azlactone ir.ten-nediate.

uia

k, > ka at the higher pH values. Thïs obervation

is not due to an increase in K, as the pH is lowered from 5.5 (where a transïent is observed) to 5.0 as ïndicated by the data of Hirohara et al. ]4] _ They found the K, of N-acetylL-tryp+&phan ethyl ester to be invariant from pH 5 to 6. With a pK - 7 for the a-amino group of L-tyrosine ethyl ester, there would be no significant change in the fraction protonated at any time between pH 5 (99%) and 5.5 (97%). Comparison of the steady-state data for Ltyrosine ethyl and -methyl esters from the present study with that lor ktyrosine p-nitrophenyl ester 1311 shows vïrtual identity of keat values at pH 7 but a six-fold differente between the alkyl and aromatic esters at pH 5 (Table 1) These data indicate that RB, deacylation, ti rate-lïmiting for these substrates at pH 7 but it. is nBt totally rate-limitïng at pH 5 for the alkyl esters. Also, the ratio K,/K, = 0.6 (= kB/(k2 + k3)) suggests that k, = k, at pH 5.0, 26.5 “C and p = 0.2M. Based on steady state nucleophile competition experïments with 2M methanol, Johnson aad Stewart [41] concluded that the rate-determinïng step for Ltryptophan ethyl ester at pH 7 was acylation However, at ti% pH we observed a large tmnsient (0.360 log(volts): C, > K,) for this substrate, as well as for Lphenylalanine ethyl ester, indicating that k3 must be the rate-determming step. K. for N-acyl tyrosine esters bound fo ~dzytnot7ypsin The observed dissociation con.stan~, K,, for the N-acyl-Ltyrosïne ethyl esters used in the present ïnvestïgation do not correlate strïctly with hydrophobicity constants (ír), molar refractivities or PK, of the parent acid of the N-acyl groups (Tables 2 and 3). The K. valu= determined by stopped-flow proflavin dïsplacement euperïmenk for +he ester substrates used in thïs study

246

compare reasonably wel1 with the K,(app) values for the correspondïng amide substrates [37]_ The corresponding K, and K,(app) values for the Ncarboalkoxy and N-formyl substrates are essentielly equivalent, while the reported K, (app) values for N-acetyl and N-chloroacetyl amitie substrates are 2- to 3-fold hlgher than the K, values for the ester substrates. Companson (Table 3) of the effect of theN-acyl moiety of the substrate on the acylation step (k?) of ester hydrolysis with that for the steady-state k cat (’ kZ) of aaide hydrolysis shows that the effect is essentially identical. Assuming that none of these substrates undergoes azlactone formation dunng enzymatic hydrolysis, it appears that the effect of the substrate N-ecyl group on forrnation of the tetrahedral intermediate (thought to be rate determting for esters [LO] ) and its breakdown (thought to be rate determining for amides [42] ) is the same. k3 for hydrolysis

of N-acyl-chymotrypsms

The correlation between log k3 and the pK, of the parent acid of the N-acyl group (R’ = 0-76; Fig. 2) supports the hypothesis that the hydrogenbonding ability of the N-acyl substituent is cf primary importante in the deacylation reaction. This correlation confïrms the conclusions of prevïous workers [13, 141 and presents experimental evidente for the postiated hydrogen bond between the N-acyl amicïo hydrogen of the substrate and tbe carbonyl oxygen of serïne-214 of the enzyme, consistent ucith X-r~y crystilography [15]_ Other data in support of this hypothesis are provided from use of substrates in which the -NHgroup is missing (i.e., fl-phenyl propionate esters [43] or replaced with an 0 (phenyl-lactic and acetyl-phenyllachc acid esters [44] or by a methylene group 138:. Devïations from lineanty (most pronomcecï for N-mesyl- and N-carbomethoxy tyrosine ethyl ester) may be due to other factors important at the enzyme-substrate lotus, as discussed below for the acylation step.

Acylation

of a-chymotrypsin

by tyrosine

esters

The data for k,, the acylation rate constant, do not correlate at al1 with the pK, of the parent acid of the N-acyl moieties. If the acylation rates were to vary as a function of the hydrogen-bondïng ability of the N-acyl substituent, the N-methanesulfonyl and unacylated substrates would be expected to have by far the highest k, values. Instead, these substrates have k2 values which are half that of N-acetyl-L-tyrosine ethyl ester. Also, a mechanism which involves transition statestabïlïzation of the acvlation reaction by a hydrogen bond between the N-acyl amido hydrogen of the substrate and the carbonyl oxygen of serine-214 of the enzyme would predict that the Nformyl substrate should react approximately 50% faster than the N-acetyl substrate; OUTdata show that the N-acetyl substrate reacts four times faster than the N-formyl substrate. Therefore, it is apparent that some function of the N-acyl moiety of the substrate other than hydrogen-bondïng is of more importante in the acylation step of the reaction.

Comparïson of the acylation rate constants wïth the molar reticfiety of the N-acyZ group shows no strong correlation. For example, the carbo-. methoxy substrak bas a higher mok re&ctivity than the N-acetyl substrak but a four-fold lower k2_ Foster and Neimann [S] preq.ïoujly co~~~lated acylation rakes obtined by steady-state studies for N-acyl-Ltyrosinamides wïth the respective N-acyl group molar refractitity; however, their study did not include the carbomethoxy substrateThe relative hydrophobicities of the N-acyl groups aLso fd t0 explaïn the acylation data. The most hydrophobic of the IV-acyl-L-tyrosine ethyl esters, the N-carbomethoxy substrak, has one of the lowest acylation rates. If desolvation of the N-acyl group were to be of prÏmary importante for reactivity as deduced from the study of Bell et al. [45], the N-carbomethoxy substrate wo-ald notbe espected to havestich alowrate relative to the ether substrates, nor would the four-fold differente in rates between the N-formyl and N-acetyl-L-tyrosine ethyl esters be expected since thelr IT values are essentially equivalent. Strict correlation of thc acylatlon rate data wïth abïlity of the N-acyl goup to undergo azlactone fonnation (Table 4) is difficult to determine in that the rates of hydrolysis for the un-ionized N-tosyl and the protonated glycine p-nitrophenyl esters relacive to the other compounds are not known. The enhanced reactivity of the N-chloroacetyl and N-acetyl substrates is predicted by the azlactone intermediate hypothesis, as is the depressed reactivity of the N-methanesulfonyl and unacylated substrates; ho% ver, the apparent identity of the acylation rates for the N-formyl and -N
248

methylated. These additional enzyme-substrate interactïons may play secondary roles in the deacylation step, thus permitting a hetter correlation between the hydrogen-bonding ability of the Nracyl substituent and the rate of deacylation. Thïs mterpretation of the acylation data is also sapported by studies on the a-chymotrypsincatalyzed hydrolyses of polypeptide amide and ster substrates. Addition of an amino acid to the NtermkËus of phenylalanïne amide mcreased the rate of hydrolysis (acylation rate determining) by three-fold in some cases [S] , but the effect on the rate of ester hydrolysis (deacylation rate determining) was much smaller and could even lead to a decrease in the rate [47]. This study has shown that there are dïfferences between the acylation and deacylation steps of xhymotrypsm catiysis, at least in detail. ft is not unexpwted that such differences exist, in that the force of evolution (at least as far as mechanistic efficiency is concerned) should act prïmarïly on the rate-limiting step of a reaction. In the case of =hymotrypsin actïng on natural substrates (polypeptides) this would be the acylatïon step. ït is apparent that achymotrypsin utilkes enzyme-substrate interactions distal to the site of the scissile bond in different ways for stabïlization of the transitlon

stat~

in the

acylation

and

deacylation

steps.

Acknowledgmenk

We are grateful to Roger Maehler for the binding data using N-methylchymotrypsin and to Clara Robison for typing the manuscript.

References 1 B. Zemer, R. Bond and M. L. Bender, J. Am. Chem. Sec., 86 (1964) 3674. 2 A. Williams, Biochemïstry, 9 (1970) 3 383_ 3 H. Hirohara, M L. Bender and R. S. Stark, Proc. Nat. Acad. Sci. U SP _, 71(1974) 1643. 4 H_ Hirohara, M_ Phillip and M. L. Bender, Biochemistry, 16 (1977) 1573_ 5 E. C. Lucas and M Caplow, J. Am. Chem. Sec., 94 (1972) 960. 6 J. Fastrez and A. R Ferscht, Biochemistry, 12 (1973) 1067. 7 B. Zeeberg, M. Caplow and M. Caswell, J. Am. Chem. Sec ,97 (1375j 7 346. 8 C. A. Bauer, R. C. Thompson and E. R. Blout, Biocbemïstry, 15 (1976) 1291_ 9 R. J. Fostcr and C. Nrcmann, J. Am. Chem. Sec., 77 (1355) 1886. 10 C. B. Sawyer and J. F. Kirsch, J. Am. Chem. Sec., 97 (1975) 1963. 11 M. A. Coletti-Prcvïero, C_ AxelrudeCavadore and A. Previero, FEB6 Lett., 11 (1970) 213. 12 J. de Jersey, P. Wdladan and B. Zemer, Biochemïstry, 8 (1969) 1959. 13 D. W. Ingles and J. R. Knowlq Biochem. J., 108 (1968) 561. 14 S_ G. Cohen, B_ Tore_m, V_ Vaidya and A. Ehret, J. Biel. Chem., 25X(1976) 4 722. 15 T_ A_ Steita, R. Henderson and D. M. Blow, J. Mol. Bio]., 46 (1969) 337_ 16 D. M. sc?gal, J. C. Powers, G. H. Cohen, D. R. Davïes and P. E. Wiicox, Biochemïstry, 10 (1971) 3 728. 17 D. M. Blow, C. S_ Wright, D. Kukla, A. Rüh!mann, W. Steigemann and R. V. Huber, J. Mol. Biel., 69 (1972) 137.

249

18 D. E. Ryan and R_ E. Feeney, d_ BioL Chem., 250 (1975) 843. 19 F. Bergel and G. E. Lewïa, J. Chem. Soo., (1957) 1816. 20 J_ H. Barna, R. C. bk_son, G. T_ Pickson. J. ?Zlks and V. D. PooIe. 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 00

41 42 43 44 45 46

47

J. Chem-

Sec..

(1953) 1448. E. F&her, Ber. Chem Ges., 37 (1904) 2 495. F. J_ Kézdy and E. T. Kuiser, Methods Emrymol., XIX (1970) 6. 1412. R. L. Bixler and C. Niemarm, J. Am. Chem. Sec.. Sl(l959) H. Gutfreund, Enzymes: Physical Prfncïples, Wiley-Intetience, N.Y., 1972, p_ l79C. Hans&, Acc. Chem. Res., 2 (1969) 232. T_ Pujita, J. Sawa and C. Hans&, J. Am. Chem. Sec., 86 (1964) 5 175. G_ N. wlkinson, Biochem. d ,80 (1961) 324. J_ E. Puidie and N. L. Benoitoa, Can. J. Biochem., 48 (1970) 1058_ A. Hbnoe, K. G. Brandt, R. J. DeSa and G. P. Hem, J. Biel. Chem., 244 (1969) 3 463. R Henderson. Biochem. J., 124 (1971) 13. F. J. KGzdy, S. P. Jindd and M. L. Bender, J. BioI. Chem., 247 (1972) 5 746. C. Hansch, A. Leo, S. H. Unger, K. H. Kim, P. Nikaïtani and E. J. Leïn, J. Med.Cbcm., 16 (1973) 1207. W_ P. Jencks and R. J_ Regerxtem, in H_ A. Sober (ed.), CRC Handboek of Biochemktry. Chemicd Rubber Co., Cleveland. 2nd edn.. 1970, J-187_ R. S. Molday and R. G. Kallen, J. Aro. Chem. Sec., 94 (1972) 6 739. M. Schemblatt and Y. Rahauün, J_ Chem. Sec., Perkin Trans. LI, (i975) 764_ J_ L. Marini and M_ Caplow. d. Am. Chem. Sec.. 93 (1971) 5 560. M YosbünotoandC. Haruzh, J. Crg. Chem., 41(1976) 2 265. G. Heb and C_ Niernann, Proc. Nat. Acad. Sci. USA_, 47 (1961) 1341. F. J. KQzdy, A. Thomsan ZUIL M. L. Bender, J. Am. Chem. Sec ,89 (1967) 1004. M. Badanazky anä M. A. Ondetci, Pepfide Syn~&&, fnteracience, N.Y., 1566, p_ 137. P. E. Jobnaon and J_ A. Stewart, Arch. Biocbem. Biophys., 149 (1972) 295. M. H. 0’Lear-y and M. D_ Kluetz, J. Am. Chem. Sec., 92 (1970) 6 089; 94 (1972) 3 585. K. J. Laidler and L. M. Earnard, Trans. Faraday Sac., 52 (1956) 497. S_ G. Cohen 2nd S_ Y. Weinstein, J. Am. Chem. Sac., U6 (1964) 5 326 R. P. Bel], J. E_ Crïtchlow and M. I. Page, J. Chem. Sec., Perkin T-. II, (1974) 66. M. Caplmv and C. HarPer, J. Am. Chem. Sec., 94 (1972) 6 508. S. A. Bizzozero, W. K. Baurnann and H. DutIer, Eur. J. Biochem., 58 (1975) 167_