Reaction of acyl carbazates with proteolytic enzymes

BIOCHEMICAL

Vol. 67, No. 2, 1975

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REACTIONOF ACYL CARBAZATES WITH PROTEOLYTIC ENZYMES JAMESC. POWERS* and DAVID L. CARROLL School of Chemistry, Georgia Institute of Technology Atlanta, Georgia 30332 Received

September

15,

1975

SUMMARY Acyl carbazates (1) with appropriate substituents are analogs of acyl amino acids in which the cr-methine group has been replaced by a nitrogen atom. The synthesis of three new carbazates Ac-Ala-Bzc-ONp, Ac-Ala-Met-ONp and Z-Ala-AlaPro-Met-ONp(Bzc= -NHN(CHC H )CO-, Met= -NHN(CH3)CO-)was accomplished. The reaction of chymotrypsin, ' &&ilisin BPN', and elastase(porcineand human leukocyte) with the carbazates resulted either in a spectrophotometric burst of Enitrophenol or no reaction depending on the specificity of the enzyme. Carbazyl chymotrypsin A crystals were isomorphous with the native protein. Acyl carbazates shouldofind utility as inhibitors and active site titrants for serine proteases. Carbazates (1) with the appropriate

substituents

are analogs of amino acids

in which the cl-methine group has been replaced by a nitrogen atom.

They would

thus be expected to acylate a serine protease with the appropriate in much the same fashion as simple synthetic enzymes (carbazyl

enzyme" due to the influence of the adjacent nitrogen atom.

derivative,

ward deacylation be suitable intermediate

The acylated

enzymes) should be considerably more stable toward deacylation

than a normal "acyl A similar

peptide substrates.

specificity

Ser-195 carbamyl chymotrypsin is completely stable to-

(1).

Carbazyl derivatives

for crystallographic

of serine proteases should thus

study of the structure

of the "acyl

in the mechanismof peptide bond hydrolysis

catalyzed

enzyme"

by this

class of enzymes. We now report the synthesis of several new peptide carbazates and investigation

of their

reaction with four serine proteases.

have shown that ethyl 3-acetyl-2-benzyl petitive

inhibitor

Previous workers

carbazate was a predominately com-

of chymotrypsin while the corresponding p-nitrophenyl

(2) can be used as an active site titrant

for chymotrypsin and trypsin

* To whomcorrespondence should be addressed

ester (2).

In

Vol. 67, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0 R,-NH-+ONp

RI AC

R2 BZ

AC-Ala

Bz

AC-Ala

Me

2

2 ,-4

R2 J

,S Z-Ala-Ala-Pro

addition,

a peptide inhibitor

synthesized

of

Me

renin containing

a carbazyl

residue has been

(4)

Coupling of Ac-Ala-OH' with benzylhydrazine

using the mixed anhydride method

method(5) gave a mixture of Ac-Ala-NHNHBz (6, 27%, m.p. 157.5 - 159', pmr (DMSOd6):N-CH2- 3.826) and the undesired isomeric hydrazide Ac-Ala-NH(Bz)NF2 (13%, oil,

pmr (CDC13):N-CH2-4.626) which were separated by silica

gel chromatography.

The desired isomer (5) was independently synthesized by reduction of the benzylidene derivative

Ac-Ala-NHN=CHC6H5(m.p.

prepared from Ac-Ala-NHNH2(6). in CHC13containing

(i-Pr)2NEt -

carbazate 2&(38%, m.p. 148'(d), with t-butyl

198.5 _ 200.5”)

Reaction of p-nitrophenyl gave E-nitrophenyl

2-methylcarbazate(7)

gave E-nitrophenyl

with p=nitrophenyl

dicyclohexylamine lysis

chloroformate with &I,

Coupling of Ac-Ala-OH

Deblocking with CF3 -

chloroformate

((L-Pr)2NEt

3-(Nacetyl-L-alanyl)-2-methylcarbazate&,

167(d), pmr (CF3COCF3'1.6 D20):N-CH3 3.386). salt)(8)

with t-butyl

was

3-(N-acetyl-L-alanyl)-2-methyl-

carbazate (38% m.p. 175-177', pmr (CDC13):N-CH33.076). C02H followed by reaction

which

3- (N-acetyl-L-alanyl)-2-benzyl

pmr (CDC13):N-CH2-4.826. gave t-butyl

(NaBH4, EtOH)

in CHC13)

(51%, m.p. 162-

Coupling of Z-Pro-OH (as the

2-methylcarbazate

followed by hydrogeno-

(5% Pd on C, 1 atm) and addition of methanolic HCl yielded HCl'Pro-NHN

(CH3)C02C(CH3)3(76%, m.p. 185.5(d), Z-Ala-Ala-OH by either yielded,

after

silica

(D20)

:N-CH3 3.056).

Coupling with

a mixed anhydride or dicyclohexylcarbodiimide

method

gel chromatography, Z-Ala-Ala-Pro-NHN(CH3)

CO,C(CH,), (82%, oil). nitrophenyl

pmr

chloroformate

Deblocking with CF3C02Hfollowed by reaction with E((i--Pr)2NEt

in CHC13)and silica

gel chromatography

I5tandard abbreviations are used for amino acid derivatives and peptide except for Bzc (2-benzylcarbazic acid) and Met (2-methylcarbazic acid).

640

Vol. 67, No. 2, 1975

BIOCHEMICAL

yielded p-nitrophenyl methylcarbazate2

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

3-(N-benzyloxycarbonyl-L-alanyl-L-alanyl-L-prolyl)-2-

(38%, m.p. 104-108', pmr (CDC13):N-CH33.036).

The reactions of the acyl carbazates were studied spectrophotometrically at 347.5 nm. Typically the appropriate hydrolysis,

carbazate in acetonitrile

buffer

solution.

After

measuring the slow rate of carbazate

50 - 500 ug of enzyme in buffer

mixture contained 5 or 10% acetonitrile over enzyme. The kinetics

was added to 0.5 - 2.0 ml of

was added. The final

and a 24 to 1000 fold excess of carbazate

were treated according to the following

E-S is the enzyme carbazate complex, E-S is the carbazyl nitrophenol

reaction

schemewhere

enzyme, HONpis -p-

and P is product(g). E+S

f l(s

ES

-

K2

E-S

k3

l

E+P

+ HONp In the majority and carbazate,

of the cases where reaction a burst of p-nitrophenol

(if any) turnover of the carbazyl The reactivity

was observed between the enzyme

occurred followed by an extremely slow

enzyme. The results are listed

in Table I.

of the serine proteases toward the four carbazates studied

is in general that expected on the basis of the substrate specificity individual

of the

enzymes. Elastase only reacts with the methyl carbazate Z-Ala-Ala-

Pro-Met-ONp(5) - and not with the two benzyl carbazates or the dipeptide

Elastase has been shown to require an extended peptide chain

Ac-Ala-Met-ONp(4).

and a small amino acid residue (e.g. Ala) as the P1 residue(l9) or inhibitor( city,

Subtilisin

reacts with all

rapidly

in a substrate

BPN' which has a rather broad substrate specifi-

of the carbazates.

Chymotrypsin AY as expected reacted

with both of the benzyl carbazates (1 and z),

inactivated,

carbazate

however it was also

although more slowly, by the methylcarbazate

(z).

Possibly the

binding of the extended peptide chain of 2 to the extended substrate binding region of chymotrypsin A(12) overpowers the lack of an aromatic side chain in the P1 residue. dipeptide

This would also account for the lack of reactivity

of the

carbazate -4.

The reaction

of Ac-Ala-Bzc-ONp(z)

with either

641

chymotrypsin Ay,Aa or subti-

ul bb

N

elastase

BPN'

faste 0

0 0

5.8g 5.0'

5.8h 6.Se

5.8

0.03

5.0'

0.20 faste

ka ac Isec-l)

b

-

4.6 x 10 -5

slow d -

cat ( set -l)

k

Serine

0

0

0 0

faste faste faste

faste

ac (set -1,

7.2 x 10 -5 3.1 x 10 -3

slow d 2 x 10 -gf

k cat [set -1)

0 0

0 0.27 faste 0

0

-

+ k3) which = k2k3/(k2 acetate, 5% acetonitrile.

is equal

to kg when k2 7 kg.

k2 > k3 and [Slo

faste fast1

faste faste

-

faste faste

-4 6.5 x 10

0.056

k ac (set -1 )

>> 5.

-

-3 6.1 x 10 1.1 x 10 -3

slow d

k cat (set-l)

Z-Ala-Ala-Pro-MecONp -

' Due to the small quantity of enzyme and Boc-Ala-ONp

of enzyme available, this experiment and observing the rate of p-nitrophenol

g O.lM citrate, 5% acetonitrile. h O.lM phosphate, 5% acetonitrile. was performed release.

by the addition

of carbazate

to a solution

d Slow indicates that little or no turnover of the carbazoyl enzyme was observed in 15-30 min., i.e. kcat 90% complete in the 9 sec. it took to mix enzyme and carbazate, i.e. k ac > ca. 0.2 set , f This is a true deacylation rate (k3) which was obtained by measuring the recovery of enzyme activity of the inactivated chymotrypsin after removal of the excess carbazate by gel filtration.

‘O.lM

b kcat

Proteases

k cat -1 (set )

k ac-1 ( set )

With

k

Carbazates Ac-Ala-Met-ONp

of Acyl

Ac-Ala-Bzc-ONp

Reaction

+ k3Ks) /(Ks + [Slo) which is equal to k2 in the cases where akac = ((k2 + k3) [Sl 0 The first condition is probably met in most of the experiments.

human leukocyte elastase trypsin

porcine

subtilisin

5.0' 5.88

Y

chymotrypsin

A

PE

Enzyme

AC-Bzc-ONp

TABLE I.

9 cn

1 f Fl 3

8

5

v, ?

0

5 N

2 b .Y

BIOCHEMICAL

Vol. 67, No. 2, 1975

lisin

AND BIOPHYSICAL

BPN, which had been titrated

acid sultone(gc),

RESEARCH COMMUNICATIONS

with 2-hydroxy-S-nitro-a-toluenesulfonic

was shown to give a stoichiometric

burst of p-nitrophenol.

of chymotrypsin A was isolated by quenching the Y reaction mixture to pH 3 followed by gel filtration on Sephadex G-25. ReThe carbazyl

derivative

generation of chymotryptic

(pH

7.45) were obtained.

reactions,

was followed using an Ac-Tyr-OEt assay

-6 rates (k3) at 26.0°C of 2 x 10 set -'

and deacylation set -1

activity

The observation that K2>>k3 in many of the

shows that acyl carbazates can be utilized

and sometimes specific

inhibitors

of serine proteases.

of the carbazates should find utility teases and we are currently define their

(pH 5.8) and 2 x 10s5

as active

as extremely effective In addition

site titrants

performing the appropriate kinetic

several

for serine proexperiments to

usefulness.

Reaction of crystals inactivated (native(13): derivative

crystals

of chymotrypsin A with Ac-Ala-Bzc-ONp(3)- yielded Y with unit cell dimension a=b=69.61 A0 and c=97.70 A0

a=b=69.6, c=97.7).

A crystallographic

investigation

of this

is in progress. ACKNOWLEDGEMENTS

This research was supported by National Institutes in part by NIH contract

HR-42939 and a Research Corporation grant.

thank Dr. Aaron Janoff at the State University kindly providing

of Health Grant GM-18292, a We

wish to

of NewYork at Stony Brook for

the human leukocyte elastase used in this work and Dr. Gerson

Cohen and Dr. David Davies of the NIH for the chymotrypsin AY phases, many computer programs and many helpful

discussions. REFERENCES

1. 2. 3. 4. 5. 6.

Robillard, G. T., Powers, J. C., and Wilcox, P. E. (1972) Biochem. 11_, 17731784. Kurtz, A. N. and Niemann, C. (1961) J. Am. Chem. Sot. 83, 1879-1882. Elmore, D. T. and Smyth, J. J. (1968) Biochem. J. 107, 103-107. Parry, M. J., Russell, A. B. and Szelke, M. (1972) Chemistry and Biology of Peptides, J. Meienhofer, Ed., Ann Arbor Science Publishers Inc., Ann Arbor, Mich., 541-544. Anderson, G. W., Zimmerman,J. E. and Callahan, F. M. (1967) J. Am. Chem. Sot. 89, 5012-5017. semion, I. Z. and Morawiec, J. (1964) Bull. Acad. Polon. Sci. Ser. Sci. Chem. -12, 295-298;(1964) Chem. Abst. -61 1340g.

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10. 11. 12. 13.

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AND

BIOPHYSICAL RESEARCH COMMUNICATIONS

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