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THE Joumia~ OF BIOLOGICAL CHE\IISTRY

Vol. 243, No. 7, Issue o f April 10, pp. 13161348, 1968

Printed in U.S. A.

Comparative Studies of the Enzymatic Properties of

Novo and Carlsberg Subtilisins*(Received for pnhlicittion, October 5, 1967)

,4. 0. BAREL$ AND A. T\T. GLAZER

From the Department oj Biolog ical Chemistry, University oj Calijoynia at Los Angeles School of Medicine, Los

Angeles, CalQwnia 90024

SUMMARY

The activit ies of Carlsberg and Novo subtilisins towards anumber of N-acetylamino acid esters and amino acid esters

have been examined. The enzymes were also compared

with respect to their eff iciency in catalyzing aminblysis reac-

tions, their rates of inactivation by certain aromatic sulfonyl

halides, and the rates of deacylation of their N-frans-cin-

namoyl derivatives. The enzymes were found to be quali-

fafively indistinguishable from the standpoint of substrate

specificity. However, significant quanf ifaf ive differences

were observed. The results obtained suggest that the

substrate-binding site of Carlsberg subtilisin is less polar

than that of the Novo enzyme, and that the groups in the

cataly tic site of Carlsberg subtilisin show a somewhat

higher reactivit y than the corresponding groups in Novo

subtilisin.

The subtilisins are a group o f alkaline proteinases originat,ing

from different strains of Bacillus subtilis. Three enzymes be-

longing to this group were isolated in pure form from the Carls-

berg (l), Novo (a), and 13PN’ (3) strains of t’his organism.

Analysis of t,he complete amino acid sequence of these prot,eins

by Smith etal. (4) has revealed that Carlsberg subtilisin diff ers

from BPX” subtilis in in over 80 positions. The enzymes ob-

t,ained from the Novo and UPN’ strains appear to be identical

(5).’ From the sequence data o f Smith et al. (4), there is lit,tledoubt that Carlsberg and Xovo subtilis ins either originated from

a common ancestral protein, or that one of these enzymes is a

precursor of t,he ot,her. The limited data available on the

enzymat,ic properties of the subtilisins show these enzymes to be

qualitatively alike. Both Clarlsberg and Novo subtilis ins exhibit

* This investigation has been aided by Grant GM 11061 from

the Sational Institutes of Health, United S tates Public Health

Service.

1 Recipient of a Fulbright Travel Grant. On leave of absence

from the Lahoratoire de Chimia Gdndrnle, Facnlti: des Science s,

Universitt5 Libre de Brnxelles, Belgium.

* S. A. Olaitan, R. J. &Lange, and E. I,. Smit)h, in preparation.

peptidase and e&erase activities (2, 6) and have very similar pH

profiles (6). Roth enzymes cabalgzc transesberification reactions

with virtually identical effi ciency (7). Last, both enzymes areinhibited by diisopropyl fluorophosphate (8, 9). Quantitative

differences were observed in the rate of cleavage of ester sub-

strat’es by Carlsberg and Novo subtilisins (2, 6).

This paper presents a continuation of the studies on the

structural and enzymatic properties of the subtilisins being

performed in this laboratory. The act ivi ty o f Carlsberg and

Novo subtilisins on a number of N-acetylamino acid esters and

amino acid esters has been examined. The enzymes have also

been compared with respect to their effi ciency in catalyzing

aminolysis reactions, and wit,h respect to their rate of inact,ivation

by various chemical inhibitors. These studies show that the

differences in primary amino acid sequence of Carlsberg and

Novo subtilisins have result,ed in significant differences betweenthe enzymatic properties exhibit,ed by these two enzymes. It

is now generally accepted that the highly specific pancreat’ic

proteases-trypsin, chymotrypsin , and elastase-represent prod-

ucts of evolution of a single anc&ral protein (10). The sub-

tilisins may be regarded as undergoing a similar evolutionary

process but are, as yet , lrss clearly different,ia ted in t,erms of

substrate specificity.

EXPERIMENTAL PROCEDURE

Carlsberg subt’ilisin (cryst,alline alcalase), Batch 50624, and

crystalline bact,erial proteinase Nova, dialyzed and lyophilized,

Batch 60, were obtained from ru’ovo Indust,ries, Copenhagen.

hct’ive site titrations (11) with A-lra,ns-cinnamoylimidazole at

pH 6.0 and 7.0, showed, respectively, 66 and 88y0 active enzyme

in the Novo and Carlsberg preparations. In agreement with

these active site titrations, the enzyme preparations were found

by dialysis to contain 30y0 (Kovo) and 9%; (Carlsberg) low

molecular weight) peptide mat,erial, presumably arising from

autolys is. The protein concentrations were detcrmincd spectro-

photomet’rically with t’he use of an Et”,,, (278 mp) value of 11.7

for Nova (12), and an Eit,,, (280 mp) value of 8.6 for Carlsberg

subtilisin (1). In most of the st’udies described below, dialyzed

enzyme preparations were used (6). When undialyzed solutions

1344

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Issue of April 10, lSci8 A. 0. Rare1 and A. N. Glam 1315

were used, the active enzyme concentration was determined by

titration wit,h AT-trans-cinnamoylimidazole. L-Leucine benzyl

ester hydrochloride, Lot LE12; glycgl-L-phenylalanyl-L-alanine,

Lot G-Phdl I ; and glycyl-L-phenylalallyl-L-phea3-lalanine, Lot

G-PhPhl, were obtained from New England Xucblear. L-

Rlanine benzyl ester hydrochloride, Lot 303.Gl709; L-phenyl-

alanine benzyl ester hydrochloride, Lot 2414.R3802; L-tyrosine

ethyl ester hydrochloride, Lot M2062; glycine ethyl ester hydro-

chloride, Lot HX1218; L-phenylalanine ethyl ester hydrochloride,

Lot N1279; n-alanine methyl est’er hydrochloride, Lot M1513;

and ~-trans-cinnamoy~nli~~azole, Lot 52236, were obtained from

Mann. Glycine benzyl ester hydrochloride was a gift from

Dr. E. L. Smith. Glycylglycinc amyl ester hydrochloride was

synthesized as previously described (13). All other peptides

and peptide derivatives were obtained from Cgclo. X11 of these

compounds were fomld t’o be pure as judged by paper chromatog-

raphy in I-butanol-acetic acid-water (4: 1: 5, by volume) and,

whenever appropriate, by paper electrophoresis at pH 1.9 and

4.7. Phenylmethanesulfonyl fluoride, Lot’ 44982, was purchasedfrom Calbiochcm. 4,4’-Riphenylenedisulfonyl chloride was an

Aldrich preparation, and I-dimethylaminonaphthalene-5-sulfonyl

chloride Lot 02-1751-9, was obtained from Pierce. Spectroqual-

it y grade p-dioxanc was obtained from Matheson, Coleman, and

Bell, and was redistilled and kept at -20”. All other chemicals

used were of reagent’ grade.

Determination oj” Esterase Act ivi ty

The rates of substrate hydrolysis were determined with t’he

aid of a Radiometer model TTTIC pH-stat as prcviouslg

described (6). The volume of t,he reaction mixtures was 5.0 or

6.1 ml, and the titrations were performed at pH 6.1, 7.0, and 8.0

at 37” with 0.025 to 0.1 N NaOH as titrating agent. So buffers

were used, and all solutions were made 0.1 M in KCl.

Hydrolysis of Peptides

The reaction mixtures contained 1.0 pmole of the different

peptides in 0.2 ml o f 0.1 M n’aHC03, pH 8.0, and 0.3 mg of

enzyme (approximately 0.01 pmole). After incubation at 37”

for 3 hours, the reaction was stopped by adding a small aliquot

of 4 N acetic acid. Appropriate aliquots of the acidified mixtures

were subjected to one-dimensional descending chromatography

on Whatman P;o. 3MM paper in I-butanol-acetic acid-water

(200:30:75, by volume) for 15 hours.

Kinetics of Aminolysis Reaction

The hydrolysis of L-leucine benzyl ester (0.025 M) by Novosubtilisin and by Carlsberg subtilisin in the presence of glycyl-

glycine ethyl ester (0.25 M) or glycylglycine amyl ester (0.25 M)

was followed at pH 7.0 and 37” in the pH-stat. The final

concentration of enzyme in both cases was 0.05 mg per ml. All

solutions contained 0.1 M KCl.

The kinetics of aminolysis was followed by removing aliquots

of the reaction mixture at suitable time intervals and adding

these to an equal volume of N acetic acid t’o give a final pH be-

tween 3 and 4, thus stopping the reaction. The ethyl and amyl

esters of L-leucy lglycylg lycine could be separated from L-leucine,

L-leucine benzyl ester, and glycy lglycine ethyl or amyl esters by

high voltage electrophoresis at pH 4.7 on Whatman No. 3MM

paper at 50 volt s per cm for periods ranging from 30 to 45 min.

Several standards of different concentrations of n-leucine, L-

lencine benzyl ester, and L-leucylglycylglycine were included on

each electrophoretogram. ,It the colic-lnsioii of electrophoresis,

the papers were dried for 30 min at’ 60”. The quantities of the

various components wcrc then determilled with the cadmium-

ninhydrin reagent o f dtfield and Louis (14) as previous ly de-

scribed (7).Kinetics of Deacylation of lY’-trans.Cinnamoyl Enzymes

The turnover (or&ant, JZcat,was determined under the condi-

tions of the normality titrations according to Bender etnl. (15).

kcat (= lc3 in this instance, since k2 >> ka) has been calculated at

pH 6.0 and 7.0 by dividing the velocity of the steady state reac-

tion by the enzyme concentrat’ion dctcrmined from the burst.

Kinetics of Inactivation with Aromatic Sulfony l IIalides

Phenylmetkanesulfonyl Fluoride and 4,4’-Biphenylenedisulfonyl

Chloride-In a typica l experiment,, two ident’ical reaction mix-

tures containing 1 ml of 0.1 M ‘Iris-HCl buffer at pH 8.0, 0.01

M CaC&, and 31.5 mpmoles of subtilisin n’ovo or Carlsberg were

kept in an ice bath. An aliquot of 5 ,uI of acetone or l-propanolwas added to the reference mixture; 5 ~1 (44.3 mpmoles) of 4,4’-

biphenylenedisulfonyl chloride in acetone, or 5 ,uI (46.5 m~moles)

of phenyhnethanesulfonyl fluoride in l-propanol was added to

the appropriate reaction mixture. The kinetics of inactivation

was followed by removing aliquots of the reaction mixtures at

suitable intervals and assaying the remaining esteratic act) ivity

with 0.05 M benzoyl-L-argininc ethyl ester as substrate at pH 8.0

and 37”. The ext,ent of inact,ivation in the reference solution due

to autolysis and denaturation was always less than 10 “;O after 3

hours of incubation.

Dansy12 Chloride-The reaction mixtures (Bontained 36 m~molrs

of protein dissolved in 1.0 ml of 0.1 M Tris-HCl buffer containing

0.01 M CaCIZ at pH 8.0 and 24”. Dansyl chloride, 750 mpmoles

in 100 ~1 of dioxane, was then added. -1 parallel incubation of a

control mixture, from which only dansyl chloride had been ex-

cluded, was also carried out, . The use of a high molar ratio of

dansyl chloride to protein was necessitated by the very rapid

hydrolysis of the reagent. The progress of inactivation was

followed as described above. The reaction was terminated by

dialysis of the reaction mixture, followed by passage through

Sephadex G-15, and the amount of dansyl cova lently bound to

the protein was estimated by using an EM value of 3300 for the

absorption of the dansyl derivat ive at 325 nm (16).

RPSULTS L

Hydro lysis of N-Acetylamino Acid Esters-The kinetic param

eters for the hydrolysis of a number of A-acetylamino acidesters by the two subtilisins are given in Table I. The Carlsberg

enzyme showed significantly higher V,,, ,, values wit)h the aro-

matic amino acid est,ers. However, with the branched chain

aliphatic substrate, Nv-acetyl-n-valine methyl ester, similar VI,,,,

values were obtained with bot,h enzymes. No hydrolysis of

AT-acetyl-n-tyrosine ethyl est,er was observed with either enzyme,

even at high concentration (0.18 mg per ml). Indeed, this

n-compound was an inhibitor of the hydrolysis of N-acrtyl-L-

tyrosine ethyl ester. The inhibition appeared to be of the

mixed type . In this connection, it may be noted that Morihara

(17) has recently shown that subtilisin RPN’ did not attack

peptide or amide bonds involving t,hc carboxyl group of nleucine

or n-phenylalanine.

2 Theabbreviationusedis: dansyl, I-dirnei hylaminonaphthalen~-5.sulfonyl.

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1346 Comparison of Novo and Cadsberg Subtilisins Vol. 243, No. 7

TllRLE 1 TABLE I\-

Kinetic constants of subtilisin-catalyzed hydrolysis of N-acetyl+ Hydrolysis of some peptide substrates by subtili sins

amino acid esters The reaction mixtures contained 1.0 pmole of substrat,e in 0.2

The reaction mixtures contained initially 0.005 to 0.025 M

substrate in 5 ml of 0.1 M KC1 containing 8yo p-dioxane by volume.

The subtilis in concentration was 0.2 to 1.5 pg per ml. Titration

was performed at pH 8.0 and 37” with 0.0125 and 0.025 N base as

titrating agent.

ml of 0.1 M NaHC03 at pH 8.0, and 0.3 mg of enzyme. Incuba-

tion was performed for 3 hours at 37”. ++‘+, about 507, hy-

drolysis; +, about 5% hydrolysis; -, no hydrolysis.

Substrate Nova Carlsberg Products

Substrate

Nova Carlsberg L-Tyr-L-Tyr-L-Tyr +++ +++ L-Tyr-L-Tyr, L-Tyr

L-Tyr-L-Tyr-CONHe +++ +++ L-Tyr-L-Tyr

Gly--L-Phe+1,.Phe + + Gly-L-Phe, z-Phe

Gly-L-Phe-L-Ala + + Gly-L-Phe, L-Ala

Gly-L-Phe - -

Gly-Gly-CONS - -

M

LV-Acetyl-L -tyrosine methyl ester. 0.09

AT-Acetyl-L-tyrosine ethyl esterb.. 0.07

N-Acetyl-L-tryptophan methyl ester. 0.09

N-Acetyl-L-phenylalanine methyl ester 0.06

N-Acetyl-L-v aline methyl ester. 0.28

vlxmxa

SIG-I

1560

731

415

415

28

vrmxa_-

St?-’

1930

1316

820

765

23

a Calculated per mole of enzyme, based on a molecular weightof 27,600.

b Data from Reference 6.

TABLE II

Ueacylation rafe constant for N-trans.cinnamoyl subtili sins

The reaction m ixture contained 1.44 X 1OW M substrate in 3.2

ml of 0.025 M phosphate buffer at pH 6.0 and 7.0, 0.1 M KCl, and

3.2o/o (v/v) acetonitrile. The active enzyme concentration was

0.4 to 2.4 X 1O-5 M.

DH I ka for NovaI

ka for Carlsberg

103 SIG-1 103 see-1

6.0 2.4 zt 0.3 6.3 rt 0.9

7.0 12 (14.5)” 60

a Data of Bender et al. (15).

TABLE III

Hydrolysis of amino acid esters by AJovo and Carlsberg subtilis ins

The reaction mixtllres contained initially the different ester

substrnt,es, at the concentrations indicated, in 5 ml of 0.1 M KC1

solution containing sltbtili sin at concentration of 0.01 to 0.1 mg

per ml. The assays were performed at pH 6.1 and 37” with 0.025

to 0.1 N NaOH in the pH-stat. The initial velocity values given

below were determin ed from the apparent zero order curves ob-

tained for the first, 5yo hydrolysis as calculated from the base up-

take.

Substrate

L-Tyrosine ethyl ester.

I,-l’henylalanine ethyl es-

ter....................

L-Alanine methyl ester.

Glycine ethyl ester.

L-Phenylalanine berrzyl

ester. ..__....__.._....

L-Leucine benzyl ester.

L-Alanine benzyl ester.

Glycine benzyl ester..

cSubstrate

0ncentratL

M

0.05

0.05

0.05

0.05

0.0075

0.05

0.05

0.05

-

3n

-.

Velocity

NOW Carlsberg

meq/min/ml/mgpotein/?d

0.0372 0.0232 1.6

0.0049

0.0018

0.0002

0.0050

0.0015

0.0002

0.0134

0.0555

0.0132

0.0028

1.0

1.2

1.0

0.0292

0.0335

0.0530

0.0031

2.2

0.6

4.0

1.1

-

acFo;; of

relative toCarlsberg

Deacylation Rates for IV-trans-Cinnamoyl Enzymes-Re-

sults shown in Table I indicated that Carlsberg subtilis in dis-

plays a significantly higher rate of deacylation with aromatic

substrates than the Novo enzyme. In order to measure k3directly, the rate constant fo r deacylation of N-trans-cinnamoyl

Novo and Carlsberg subtil isins was determined at pH 6.0 and

7.0. ka has previous ly been reported for N-trans-cinnamoyl

Novo subtilisin at pH 7.0 by Bender et al. (15). At both pH

values, the deacylation of N-trans-cinnamoyl Carlsberg subtilisin

is much faster than that observed with Novo subtilisin (Table II).

Rate of Hydro lysis of Free Amino Acid Esters--The kinetic

measurements with these substrates were made at pH 6.1, since

at higher pH values the rate of spontaneous hydro lysis, par-

ticularly of the benzyl esters, became considerable. Work at

higher pH values was also avoided because of the possibility of

aminolysis (see below). It will be readily seen from the data

listed in Table III t,hat, in striking contrast to the situation with

the N-acetylamino acid esters discussed above, Novo subtilisin

hydrolyzes free amino acid esters at least as readily as Carlsberg

subtilisin. The major difference between the two classes of

substrates is one of polarity. The fac t that replacement of the

N-acetylamino group by the more polar free a-amino group

reduces the catalytic eff iciency of Carlsberg subtilisin relative to

that of the Novo enzyme suggests that, of the two subtilisins,

the Carlsberg enzyme has the more nonpolar substrate-binding

site. This point is discussed further below.

Of the eight free amino acid esters examined, L-leucine benzyl

ester was the only one to be hydrolyzed faster by the Carlsberg

enzyme at pH 6.1.3 In addition to the substrates listed in Table

III, L-glut amic acid a-benzyl ester was readily hydrolyzed by

both enzymes.Hydrolysis of Small Peptides-Preliminary results with a few

di- and tripeptides, summarized in Table IV, indicate that the

subtilisins readily cleave bonds COOH-terminal to tyrosine and

phenylalanine, but that dipeptides, e.g. tyrosyltyrosine, are

resistant to further hydrolysis. Hydrolysis at tyrosine was

found to be considerably faster t’han at phenylalanine. Careful

examination of the reaction mixtures by electrophoresis and

chromatography indicated that transpeptidation did not occur

with the substrates listed in Table IV under the conditions

studied.

3 At pH 6.05 at 37” in 0.1 hf KCl, L-leucine benzyl ester gave an

apparent K, of 0.16 M and an apparent V,,, of 64 set-’ with Novo-

subtilisin , and an apparent K, of 0.30 M and an apparent V,,,,

of 248 set-1 with Carlsberg snbtilisin .

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Issue of April 10, 1968 A. 0. Bare1 and A. hr. Glaze? 1347

Aminolysis Reactions--Most of the well charact,erised prote-

olytic enzymes have been shown to catalyze transfer reactions to

amine acceptors (18). Such reactions have not been previously

demonstrated for the subtilisins. The hydrolysis of L-leucine

benzyl ester by the subtilisins was studied in the presence of a

variety of amine acceptors. It was found that both subtilisins

catalyzed reactions of the type

L-Leucine benzyl ester + glycy lglyc ine n-amyl ester eL-leucylglycy lglycine n-amyl ester + benzyl alcohol

The resulting tripeptide ester was cleaved further to the free

tripeptide by the subtilisins, but this reaction was very slow

under the conditions chosen. L-Leucy lglycylg lycine was not

hydrolyzed further. The kinetics o f the aminolysis reaction

was examined in detail in mixtures containing L-leucine benzyl

ester in the presence of 0.25 M glycylglycine ethyl or amyl ester

(see Fig. I, for example). At a molar ratio of the acceptor to

water of approximately 1:200, the ratio of the initial rates of

aminolysis to hydro lysis, at pH 7.0, ranged from 1: 10 to 1:20.The amino group o f the acceptor is only partially ionized at this

pH. Thus, like other proteolytic enzymes, the subtilisins are

efficient cata lysts of aminolysis reactions. Under the conditions

studied (see Fig. l), the initial rate of aminolysis relative to

hydrolysis in the presence of Nova subtilisin appeared to be

about twice as fas t as that obtained with Carlsberg subtilisin.

Inactivation of Xubtilis ins with Aromatic Sulfonyl Halides-

Phenylmethanesulfonyl fluoride has been shown to be a stoichio-

metric inhibitor of Novo subtilisin and to react with the serine

residue at the active site (19, 20). Comparison of the rates of

inactivation of the two subtilisins by this reagent (Table V)

showed that the Carlsberg enzyme reacted 3 to 4 times faster

than the Novo enzyme under the same conditions. To extend

this observation, the rate of inactivation of the subt’ilisins with

t,wo other aromatic sulfonyl halides was st’udied. Both 4,4’-

biphenylenedisulfonyl chloride and dansyl chloride appear to

react principaliy at the active site of the subtilisins. The loss

,

0 IO 20 30 40 50 60 70

TIME (IN MINUTES)

F IG . 1. Time course of the hydrolysis of L-leucine benzyl esterby Novo and Carlsberg subitilisins. The reactions contained L-

leucine benzyl ester (0.025 M), glycy lglyc ine amyl ester (0.25 M) ,

and enzyme (0.05 mg per ml) in 0.1 M KC1 at 37”. The pH wasmaintained at 7.0 by means of a pH-stat. Filled symbols (A,

w, 0) refer to the mixtllre containing Carlsberg suhtllisin; opensymbols (A, 0, O), to that containing Xovo subtilisin.

TABLE V

Kinetics of reaction of subtili sins with aromatic su lfonyl halides

For experimental details, see the text.

Halide

I’henylmethanesulfonyl flno-

ride.4,4’-Biphenylenedisulfonyl

chloride.

Dansyl chloride..

/

Molar ratio ofreagent to protein

1.5 3.5 1

1.4 160 129

21 25 19

a t+ s the time reyllired for the inhibition of 507, of the esteratic

act ivi ty of the subtilisirrs towards A-benzoyl-L-arginine ethylester at pH 8.0 and 37”.

in esteratic act ivi ty in the presence o f these reagents could be

very closely correlated with disappearance of active sites as

determined by Gtration with N-trans-cinnamoylimidazole or

phenylmethanesulfony l fluoride. With these reagents also, the

Carlsberg enzyme showed more rapid rates of inactivation than

Novo subtilisin (Table V), although the differences were not as

pronounced as with phenylmethanesulfonyl fluoride. As with

chymotrypsin (16), the reaction with dansyl chloride is not

entirely limited to the active site. With Novo subtilisin, for

example, incorporation of 1.2 moles of dansyl into the prot,ein is

associated with only 60% inactivation. However, reaction at

the additional sites appears to have little ef fec t on the act ivi ty.

DISCUSSION

Both subtilisins display a broad specif icity towards amino acidester substrates, as might be expected from their action on

proteins (21). The enzymes showed considerably higher activity

towards the esters of the aromatic amino acids than those of the

aliphatic ones. This is consistent with the earlier observations

on the inhibition of these enzymes by compounds such as indole

and hydrocinnamate (6). The V,,,, values obtained with

Carlsberg subtilisin with aromatic N-acetylamino acid esters were

significantly higher than those obtained with Novo (Table I).

This suggests that the deacylation of Carlsberg subtilisin

proceeds at a faster rate than that of Novo. The observed rates

of deacylntion of N-trans-cinnamoyl Carlsberg and Novo sub-

tilisins are certainly consistent with this suggestion. Little

difference was observed in the activi ties of Novo and Carlsberg

subtilisin, towards a number of free amino acid esters. Indeed,

Carlsberg subtilisin was considerably less active towards the

benzyl esters of phenylalanine and alanine than was Novo

subtil isin. The contrast between the results obtained with t,he

N-acetylamino acid esters and free esters strongly suggests that

the substrate-binding site of Carlsberg subtilisin is less polar

than that of the Novo enzyme, and hence is more sensitive to the

presence o f the polar amino group in the free amino acid esters.

The faster reaction of Carlsberg as compared with Novo sub-

tilisin with aromatic sulfonyl halides is consistent with this view.

It is possible, however, that the observed difference in inactiva-

tion kinetics may simply reflec t a somewhat higher reactivit y of

the groups in the catalytic site of Carlsberg subtilisin rather

than a higher binding aff ini ty for these reagents.

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1348 Vol. 243, No. 7

Xovo and Carlsberg subtilisins diff er in 85 positions of t,heir

amino acid sequence (4, 5). Yet’, the comparisons betIT-een

these t\v o enzymes made in this and the preceding papers sholv

that the enzymes are qualitatively illdistinguishable from the

standpoint of enzyme spec ific ity. However, a number of

quantitative observations indicate that Carlsbcrg subtilisin sho\vsa more pronounced spec ifici ty tolvards substrates containing

aromatic amino acid side chains than is evident rr-ith Nova

subtilisin. It Tvill be of considerable interest to esamille other

aubtilisins a-it,h rcspe6t to gradations in spec ific ity.

The subtilisins may, therefore, be added to the grooving list of

proteins, e.g. cytochromes (22) and bovine and rat, ribonucleases

(23), in lvhich replacement of a third or more o f the amino acid

residues appears to produce little or no change in funct ional

properties. Clearly, in these cases, the structural features

essential to function have been conserved.

dcknowledgment-The aut,hors are indebted to Dr. Emil L.

Smith for his interest in this lvork and helpful discussions.

REFERENCES

1. Gi:x'rELIIERG, A. v., AND OWESEN, >I., Compt. Rend. Trav.

Lab. Carlsb erg, 29, 36 (1954).

2. OTTESEN, M., AND SPECTOR, A., Compt. Rend. Trav. Lab.

Cadsberg, 32, 63 (1980).

3. M.~W~BARA, H., HAGIHARA, B., SAKAI, PII., KOMAICI, T.,YONJWANI, T. , AND OIWNCXI, K., J. Biochem. (Tokyo),

45, 251 (1958).

4. SMWH, E. L., MAI~I~L~ND, F. S., KMPER , C. B., DEL.~XGE,

11. J., LANDON,M., AND EVANS , W. II., 3. Biol. Chem., 241,

5974 (1966).

5. SMITH, E. L., Abstracts Seventh International Congress of

Biochemistry, 196Y, I-1,5, p. 9.

6. GLBZER, A. S., J. Bid. Chem., 242, 433 (19G7).

7. (+LAZER, A. ,RJ.,J. Biol. Chew., 241, 635 (1966).

8. OTT ESE N, M., -4~11 SCHELLMAN, C. (I+., Coopt. Rend. Tram.

Lab. Curlsberg, 30, 157 (1957).

9. I\IATSUHARA, H., AND NISHIMURA, S., J. Biochem. (Tokyo),

45, 413, 503 (1958).

10. HARTL EY, B. S., BROFX, J. li., KAL-FFMAN, 1). L., AND SMII,-LIE, 13. B., Satwe, 207, 1157 (1965).

Il. SCHONDAUM, C. II., ZERNER, B., AND BENDER, M. L., J. Biol.

Chem., 236, 2930 (19Gl).

12. &IliTSITuARA. II.. KASPER. C. B., BROWS, n. PI/I., ANI) SMITH,

E. L., J. j3iol: C hem.. i40, 1125 (1965).

13. GLAZE;, A. K;., J. Biol. Chem., 241, 3811 (1966).

14. ATFIELD, CT. N., A SD MORRIS, C.J. 0. R., Biochem. J., 81, GO(i

(1961).

15. BENDER, M. L., BEGU-CANTON, M. L., BLAKE LEY, I<. L.,

BRUBACHEK.. L. J.. FEDER. J.. COUNTER, C. I:.. KI~ZDT-.

F., KILLHE~FER, j. V., JR., &~ARSH~LL, T. H.,'MILLER;

C. G., ROESKE, n. W., AND STOOPS, J. K., J. ATneT. Chem.

Sot., 88, 5890 (19%).

IG. HARTLIW , B. S., ASU MASSEY, V., I?iochim. Biophys. .lcta,

21, 58 (1956).

17. MORIHARA, K., Biochem. Biophys. Res. Corwmnn., 26, 656

(1967).

18. FRUTON, J. S., ill II. n'~csa~a (Editor), I'he proleins, Vol. 1,

Academ ic Press, Yew York, 1963, p. 205.

19. I'OLGAR. L.. .~ND BENDEK. 31. L., J. Amer. Ch,ew. Sot., 88,

3153 (i96Ci).

20. NEET, K. E., AND KOSHLAND, 11. X., JR., Proc. ,Vat. =1cad. Ski.

I;. S . A.. 66. 1GOti (1966).

21. TUPP Y, H.: :Ilwzatsh. Chkn. Verwantdte Tiele Anderer Wiss. ,

84, 996 (1953).

22. >1.4l%GOLIASH, E., AR‘D SnxIm, E. L., irl I-. BRYSON AND 11.

\'OGEL (Editors), Evolving genes and proteins, Academ ic

Pres s, N&v York; 1965, p. -221.

23. BEINTEMA. .J. .J.. AND (+RCDEIL. h2.. Albstracts Seventh Inter-

national’Congrkss 0s Kiochernktry,’ 196Y, A-117. b  y  g u e s  t   , onM ar  c h 2  9  ,2  0 1 2 

www. j   b  c . or  g

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