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Inactivation of the Trypsin-Kallikrein Inhibitor (Kunitz) by Cleavage of the Dipeptide Ala 16ЧArg 17 in the Reactive Site.

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The Arg-I 5 inhibitor contains a single-stranded peptide chain
and thus corresponds to a protein with the mutation Lys 15
+Arg 15. We have also found this substitution as a natural
mutation in the homologous isoinhibitors from snails (Helix
pomatia)[6J.
Received: June 18, 1974 [Z 68c IE]
revised: July 16, 1974
German version: Angew. Chem. 86,704 (1974)
. _ _ _
[l] W R . Finkenstadt and M . Laskowski, Jr., J. Biol. Chem. 240, PC 962
(1965); M . Laskowski, Jr. and R . W Sealock in P. D. Eoyer: The Enzymes.
Vol. 111, Academic Press, New York 1971, p. 375.
[2] R . W Sealock and M . Laskowski, Jr.. Biochemistry 8, 3703 (1969).
[3] 7: R. Leary and M . Laskowski, Jr., Federat. Proc. (1973); D. Kowalski,
T R . Leary, R . E . McKee, R. W Sealock, D . Wang, and M . Laskowski, Jr. in
H . Fritz, H . Tschesche, L. J. Greene, and E . Truscheit: Proteinase Inhibitors,
2nd International Research Conference-Bayer Symposium V. Springer,
Berlin. in press.
[4] H. Jering and H . Tscheschr. Angew Chem. 86, 702 (1974); Angew. Chem.
internat. Edit. 13. 660 (1974).
[S] H . Fritz, I . Trautschold, and E . Werle in H . U . Bergmeyer: Methoden
der enzymatischen Analyse. Verlag Chemie, Weinheim 1970. 2. Edit., Vol.
1 , p. I01 1 N.
[6] H . Tschesche and T Dietl, Eur. J. Biochem. 30, 560 (1972); 7: Dietl
and H . Tschesche in [3].
Inactivation of the try pin-Kallikrein Inhibitor (Kunitz)
by Cleavage of the Dipeptide Ala 16-Arg 17 in the
Reactive S i t e [ * * ]
By Helmut Jering and Harald Tschesche[*]
Cleavage of the residue P I (Lys 15) from the reactive site
of the modified trypsin-kallikrein inhibitor I* (Kunitz)"' inactivates the inhibitorl2I. The question arises, whether removal
of the residue Pi (Ala 16) likewise inactivates the inhibitor,
even though the formation of an acyl-enzyme complex at
PI is still possible as an intermediate step enroute to the
stable enzyme-inhibitor complex G31.
Cleavage of the residue Pi (Ala 16) from I* meets with difficulties, since Edman degradation as used for soybean trypsin
inhibitor ( K ~ n i t z ) [is~ ]not applicable owing to inactivation
of the inhibitor (NE-derivatization),and several aminopeptidases [leucine aminopeptidase from bovine eye lenses (M.W.
300000'51),aminopeptidase M, etc.] d o not attack I*. Because
of its small molecular weight (19000r61)aminopeptidase K
is effective and cleaves the residues Pi and P; (Ala 16 and
Arg 17) within 20 minutes (0.5 pmol I*, 0.2 mol-% aminopeptidase K in 1.5 ml 0.025 M diethyl barbiturate buffer, pH = 8.3)
(Scheme 1).
p2
P: Pi Pi P;
PI
0
-Cys-Lys-COOo
L
S
-
H3N-Ala-Arg-Ile-Ile
5
Aminopeptidase K
S
pz
Pj P;
PI
0
-cys-Lys-coo~
I
H3N-Ile-Ile
r
D e s - ( A l a 16, A r g 1 7 ) - I *
Scheme I
The reaction can be so steered that up to 9 5 % modified
des-(Ala 16, *Arg 17tinhibitor is formed. Prolonged enzyme
reaction cleaves also Ile 18.-The reaction then comes to
a standstill.
The modified des(Ala.16, Arg 17hinhibitor could be separated
from the enzyme by molecular sieve filtration on Sephadex
G-50 in 0.1 M acetic acid and was purified by ion-exchange
chromatography on CM-Sephadex C-25 in 0.01 M sodium
borate buffer, pH = 8.0 (linear NaCl gradient of 0 to 0.3 M).
The homogeneous material is characterized by its amino acid
composition. Native inhibitor is not attacked at the N-terminus (Arg-Pro-Asp-Phe-) by aminopeptidase K.
After cleavage of the residues Pi and P;, I* is inactive towards
trypsin in the usual test[71, i. e. the association constant K,,,
is reduced by about 8 to 10 orders of magnitude. Whether
the acyl-enzyme complex still forms as a n intermediate is
a t present under investigation. Since Arg 17 contributes substantially to the stabilization of the complex131a drastic reduction in the association constant K,,, had to be reckoned
with; however, according to X-ray structural data for the
crystallized complex131there should still be sufficient interaction for oriented incorporation of des-(Ala 16, Arg 17)-inhibitor into the enzyme.
Received: June 18, 1974 [ Z 68d IE]
revised: July 16, 1974
German version. Angew. Chem. 86,705 (1974)
-___
[l] H . Jering and H. Tschesche, Angew. Chem. (16,702 (1974); Angew. Chem.
internat. Edit. 13, 660 (1974).
[2] H. Jering and H . Tschesche, Angew. Chem. 86,703 (1974); Angew. Chem.
internat. Edit. 13, 661 (1974).
[3] R . Huber, D. Kukla, W Steigemann, J . Deisenhofer, and A. Jones in
H. Fritz, H . Tschesche, L. J . Greene, and E . Truscheit: Proteinase Inhibitors,
2nd International Research Conference-Bayer Symposium V. Springer, Berlin, in press; see also H . Tschesche, Angew. Chem. 86, 21 (1974); Angew.
Chem. internat. Edit. 13, 10 (1974).
[4] D. Kowulski, T R . Learp, R. E. McKee, R . W Sealock, D. Wang, and
M . Laskowski, Jr. in H . Fritz, H . Tschesche, L. J . Greene, and E. Truscheit:
Proteinase Inhibitors, 2nd International Research Conference-Bayer Symposium V. Springer, Berlin, in press.
[5] S. R . HImmelhoch and E. A . Peterson, Biochemistry 7, 2085 (1968).
163 N . Hennrich, M . Klockow, H. D. Orth, U . Femfert, P. Cichocki, and
K . Jan), Hoppe-Seylers Z. Physiol. Chem. 354, 1339 (1973).
[7] H . Fritz, I . Trautschold, and E . Werle in H. U . Bergmeyer. Methoden
der enzymatischen Analyse, Verlag Chemie, Weinheim 1970. 2. Edit., Vol.
I , p. 1011 If.
'
Photoisomerization of Tricyclo[ 5.21.04. OJdeca-2,5,8triene ( T r i q u i n a c e n e ) [ * * l
By Dieter Bosse and Armin de Meijere"]
Thermal isomerization of diademane
leads to triquinacene (2). The reverse reaction is, however, conceivable only
as a photochemical two-step process. Because of the di-n-methane system present in the molecule (2)"' a diradical of structure (3) should be formed as the first intermediate upon
excitation by light. There then arises the interesting question
whether an intramolecular cycloaddition of (3) to yield ( I )
can occur.
With this in view we irradiated a 0.8 percent pentane solution
of (2) in a falling-film recycling apparatus at -40°C by
[*] Pnv.-Doz. Dr. H. Tschesche and Dr. H. Jering
Organisch-Chemisches Laboratorium,
Lehrstuhl fur Organische Chemie und Biochemie der
Technischen Universitat
8 Mbnchen 2, Arcisstr. 21 (Germany)
[**I This work was supported by the Deutsche Forschungsgemeinschaft.
We wish to thank Fraulein C. Frank for carrying out the amino acid analyses
Thanks are also due to Bayer AG, Wuppertal for kindly supplying us with
Trdsylol' (trypsin-kallikrein@ inhibitor (Kunitz) from bovine lung).
Angew. Chem. internat. Edit.
Vol. 13 (1974)
1 No.
10
[*] DipLChem D. Bosse and Doz. Dr. A. de Meijere
Organisch-Chemisches lnstitut der Universitat
34 Gattingen, Windausweg 2 (Germany)
['"I Presented in part at the Chemiedozententagung in Stuttgart on March
31-April 4, 1974 and at the 15th Conference on Reaction Mechanisms
1974.-This work was
at Fort Collins, Colorado (USA) on June 24-28,
supported by the Deutsche Forschungsgemeinschaft and the Fonds der
Chemischen Industrie
663
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site, cleavage, inactivation, 16чarg, inhibitors, dipeptide, trypsin, kallikrein, reactive, kunitz, ala
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