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Specificity of alkaline proteinase of Aspergillus flavus
386
BIOCHIMICA ET BIOPHYSICA ACTA
SHORT
COMMUNICATIONS
BSA 63442 Specificity of alkaline proteinase of
Aspergillus flavus
In preceding papers we reported on the preparation of an extracellular proteinase from Aspergillus flavus 1 and on the determination of the serine active center 2, and discussed the similarity between proteinases of various kinds of aspergillP -4. In this paper, the specificity of the proteinase isolated by us (cf. ref. 6) will be described and compared with the known specific(ties of the proteinases mentioned above. Cleavage of the B-chain of oxidized insulin. The B-chain (5 rag) of oxidized insulinT, s was digested by the alkaline proteinase of A. flavus 1 t h at 37 ° and pH 8.5. The molar enzyme to substrate ratio was I :IOO. Individual peptides were isolated by preparative paper chromatography in System S 1 (n-butanol-acetic acid-pyridinewater, 3o:6:2o:24, by vol.) 9 and b y high-voltage paper electrophoresi¢ ° at p H I . 9. The amino acid composition was determined by the method of SPACKMAN et al. 11. The peptides obtained are represented by horizontal lines in Fig. I. We also obtained a complex mixture of peptides in yields lower than o.o 4/m~ole, peptides which could not be purified further owing to their small amounts.
Mold rotei . . . .
2
;
;
1
~ ;
;
;
;
;
;
;
i
m
AspergilloN~ peptidase C"
2
;
o Alkaline
;
;
;
;
;
;
;
proteir~ase 13
NN~ NN2 SO3H SO3H Phe-VaL-AS~-GlU-His-Leu-Oys-G~-get-His- Leu-Vel-Glu-AIo-L• u-Tyr-Leu-Val-Cys-G~y-Glu-Arg-Gly-Phe4:~he-Tyc-Thr~O--LyS~Ala 1 2 3 4 5 6 7 8 9 10 11 12 13 14 }5 16 17 18 }9 20 21 22 23 24 25 26 27 28 29 30 Alkaline proteinase (this work)
~' ~'
?
~ 1I
? ?
1'
? t
'~
4~,2h
c~I F:S'I:IO00c
T
£'-~ / 4' 2h ~ ' _:S,1:1000
t
t 7o,E:5,1:100 h ~-4
Yield Of peptides
t i'
t
tt
? 1'
t
t
t 1'
t 0.749 0.73
0.64 0.38 0.37
0.4
0.33
0.2 0.11
0.25 0.15
0.06
0.1 0.08
Fig. I. Sites of cleavage of oxidized B-chain of insulin by alkaline proteinases from Aspergilli. The yield in #moles of each individual peptide calculated from the d a t a of amino acid analysis is given above the bar. The peptide containing Residues 5 I3 was assigned its location after determination of its N-terminal group b y the d i m e t h y l a m i n o n a p h t h a l e n e s u l f o n a t i o n technique 1",17. * Abbreviations used: Ac , a c e t y l - ; Boc-, tert.-butyloxycarbonyl : pNa, p-ifitro~tnilide; Sue , succinyl ; Z, b e n z y l o x y c a r b o n y l - ; - O E t , e t h o x y ; -OMe, m e t h o x y .
]3iochinL Biophys. ,4eta, 19,~ (I97 o) 386 388
387
SHORT COMMUNICATIONS
We investigated the cleavage under various conditions and thus found the sites of primary attack which correspond to the isolated peptides in order of their yield (cf. Fig. I). Cleavage of synthetic substrates. A volume of 200/*1 of 0.05 M trimethylamine buffer, which had been adjusted to pH 4, 5, 6, 7, 8 or 9 by acetic acid, was mixed with 20 #1 of a solution containing 0.05/*mole of substrate in water (or in 50% ethanol) and with IO/,1 of the enzyme solution. The resulting molar enzyme to substrate ratio was always 1:20. The reaction mixture was incubated 16 h at 37 °. The cleavage was studied by paper chromatography in System S1. The sites of cleavage of substrates containing a higher number of amino acid residues were established in analogous experiments carried out on a preparative scale (I/,mole of substrate at pH 8). The isolated digestion products were characterized by amino acid analysis 11. In the pH range 7-9 the following substrates * were cleaved completely: Gly-Phe-OMe
Gly-Phe-pNa
Gly-Phe-Leu
Leu-Gly-pNa
Leu-Leu pNa
Gly-Tyr OEt
Asn-Ala P r o - L e u - G l y - N H 2
4
Boc-/%Ala-Trp-Met-Asp-Phe-N H ~ Z G l y - P r o - G l y - G l y Pro-Ala
There was no cleavage of the following substrates : Glu Asn Glu-Glu-Ala Suc-Phe-pNa
Pro-Leu-Gly-NH 2 Ac-Phe-Tyr Gly-Pro-Ala
Our results obtained concerning the digestion of synthetic peptides show that the proteinase cleaves peptide bonds at the carboxyl side of phenylalanine, leucine, tyrosine, aspartic acid, and methionine. A fact deserving interest is that the proteinase also splits the bond at the carboxyl side of glycine, if the carboxyl of the adjacent phenylalanine residues is bound by a peptidic or at least pseudopeptidic residue, not, however, if the phenylalanine carboxyl is esterified. It is likely that in some cases the substitution of the N-terminal amino acid prevents the cleavage. By contrast, the elongation of peptide P r o - L e u - G l y - N H 2 at its N-terminus rendered the peptide susceptible to cleavage. The specificity of the alkaline proteinase of A. flavus, as observed with the digestion of the B-chain of oxidized insulin, strongly resembles the specificity of the mold proteinase of A. oryzae described by SANGER et al. TM (cf. Fig. I) ; it differs, however, from the specificity of aspergillopeptidase C of A. oryzae as reported by NORDWIG AND JAHN 4. The specificity of our proteinase corresponds to the specificity of the alkaline proteinase isolated from A. oryzae by MORIHARA AND TSUZUK113. These authors, however, report for their preparation a molecular weight of 52 ooo, which is different from the value of 18 ooo-19 ooo reported by other authors3, 4 and by us 1. Our results provide additional evidence of the great similarity existing between the proteinase of A. flavus and the alkaline proteinase of A. oryzae (cf. refs. I, 2). Biochim. Biophys. Acta, 198 (197 o) 386-388
388
SHORT COMMUNICATIONS
The d a t a p r e s e n t e d a b o v e show t h a t the specificity of t h e alkaline proteinase of
A. flavus is r a t h e r b r o a d . In t h e i r s t u d y on the b i n d i n g site of papain, SCHFCHTH~ AND BERGEn 14 r e p o r t e d a seven-residue b i n d i n g site of a s p e r g i l l o p e p t i d a s e B (@ ref. 3)- F o r this reason, we a r r a n g e d all the p e p t i d e s which are cleaved b y our proteinase in such a m a n n e r t h a t the a m i n o acids e q u a l l y d i s t a n t from the site of cleavage were below each other. W h e n c o m p a r i n g the n e i g h b o r h o o d of t h e cleaved bond, we did n o t find a n y obvious regularity. The low specificity of t h e p r o t e i n a s e was in good a g r e e m e n t w i t h t h e assumed m u l t i p l i c i t y of the binding site and o b v i o u s l y plays a f u n c t i o n a l role in the proteinases of lower organisms. The results of our i n v e s t i g a t i o n of the cleavage of b o t h the t3-chain of oxidized insulin a n d also the s y n t h e t i c p e p t i d e s lead us to conclude t h a t the proteinase isolated b y us falls into the g r o u p of serine proteinases of b r o a d specificity ta, a group to which also belongs, a m o n g others, subtilisin with the same serine active center 2,~s. W e wish to acknowledge the skillful technical assistance of Mrs..]. l.uke~ovfi. W e are i n d e b t e d to Mr..J. Zbro~ek a n d Miss V. Himrovfi for amino acid analyses.
Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Science, Prague (Czechoslovakia)
.l. "I'URKOV.'~ (). MIKF.g
I J. TURKOVA,O. MIKEg, 1{. (;AN(~EVAND ,1~'I.BOUI~LiK,Biochim. Biophys. Aela, 178 (I969) ioo. 2 0 . MIKE.~, J. TURKOVA, NGUYEX BAO TOAN AND F..~ORM, Biochim. Biophys. Acta, z78 11969) 3 4 5 6 7 8 9 1o I1 I2 13 14 15 16 17
II2. A. R. SUBRAMANIAN AND (;. KALNITSKY, Biochemistry, 3 11964) i 8 6 8 . A. NORDWlG AND VV. F. JAHN, European J. Biochem., 3 (1968) 519 . F. SANGER, Proc. Chem. Soc., 11963) 76. J. TURKOV• AND O. MIKEg, Abstr. 6th Federation European Biochem. Nocs. ,]leetin/~,, Madrid 1969, No. 187 . C. H. HIRS, J. Biol. Chem., 219 11959 ) 611. B. KEIL AND H. I(EILOV,~, Collection Czech. Chem. Commtzn., 29 (t91)4) 22o(i. S. G. WALEY AND J. WATSON, Biochem. J., 55 (1953) 328. Z. PRUSfK AND B. KEIL, Collection Czech. Chem. Commun., 25 (t96o) 2o49. D. H. SPACKMAN, ~V. H . STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 119o. F. SANGER, E . O. P. THOMPSON AND R. KITAI, Biochem. ,[., 59 11955) 509. |~[. MORIHARA AND H. TSUZUKI, Arch. Biochem. Biophys., 129 (1969) 620. 1. SCHECHTER AND A. BERGER, Biochemistry, 5 (1966) 337 I. H. F. NOLLER AND S. A. BERNHARD, Biochemistry, 4 11965) 1i~8. W . R. GRAY AND B. ~. HARTLEY, Biochem. J., 89 (1963) 379. J. NOVOTN') AND t;. ];RANbZK, Chem. I_isty, 62 11968) 995.
R e c e i v e d S e p t e m b e r 29th, 1969 Biochim. Biophys. ,4cta, TOS (197 o) 386 3 , ~
BIOCHIMICA ET BIOPHYSICA ACTA
SHORT
COMMUNICATIONS
BSA 63442 Specificity of alkaline proteinase of
Aspergillus flavus
In preceding papers we reported on the preparation of an extracellular proteinase from Aspergillus flavus 1 and on the determination of the serine active center 2, and discussed the similarity between proteinases of various kinds of aspergillP -4. In this paper, the specificity of the proteinase isolated by us (cf. ref. 6) will be described and compared with the known specific(ties of the proteinases mentioned above. Cleavage of the B-chain of oxidized insulin. The B-chain (5 rag) of oxidized insulinT, s was digested by the alkaline proteinase of A. flavus 1 t h at 37 ° and pH 8.5. The molar enzyme to substrate ratio was I :IOO. Individual peptides were isolated by preparative paper chromatography in System S 1 (n-butanol-acetic acid-pyridinewater, 3o:6:2o:24, by vol.) 9 and b y high-voltage paper electrophoresi¢ ° at p H I . 9. The amino acid composition was determined by the method of SPACKMAN et al. 11. The peptides obtained are represented by horizontal lines in Fig. I. We also obtained a complex mixture of peptides in yields lower than o.o 4/m~ole, peptides which could not be purified further owing to their small amounts.
Mold rotei . . . .
2
;
;
1
~ ;
;
;
;
;
;
;
i
m
AspergilloN~ peptidase C"
2
;
o Alkaline
;
;
;
;
;
;
;
proteir~ase 13
NN~ NN2 SO3H SO3H Phe-VaL-AS~-GlU-His-Leu-Oys-G~-get-His- Leu-Vel-Glu-AIo-L• u-Tyr-Leu-Val-Cys-G~y-Glu-Arg-Gly-Phe4:~he-Tyc-Thr~O--LyS~Ala 1 2 3 4 5 6 7 8 9 10 11 12 13 14 }5 16 17 18 }9 20 21 22 23 24 25 26 27 28 29 30 Alkaline proteinase (this work)
~' ~'
?
~ 1I
? ?
1'
? t
'~
4~,2h
c~I F:S'I:IO00c
T
£'-~ / 4' 2h ~ ' _:S,1:1000
t
t 7o,E:5,1:100 h ~-4
Yield Of peptides
t i'
t
tt
? 1'
t
t
t 1'
t 0.749 0.73
0.64 0.38 0.37
0.4
0.33
0.2 0.11
0.25 0.15
0.06
0.1 0.08
Fig. I. Sites of cleavage of oxidized B-chain of insulin by alkaline proteinases from Aspergilli. The yield in #moles of each individual peptide calculated from the d a t a of amino acid analysis is given above the bar. The peptide containing Residues 5 I3 was assigned its location after determination of its N-terminal group b y the d i m e t h y l a m i n o n a p h t h a l e n e s u l f o n a t i o n technique 1",17. * Abbreviations used: Ac , a c e t y l - ; Boc-, tert.-butyloxycarbonyl : pNa, p-ifitro~tnilide; Sue , succinyl ; Z, b e n z y l o x y c a r b o n y l - ; - O E t , e t h o x y ; -OMe, m e t h o x y .
]3iochinL Biophys. ,4eta, 19,~ (I97 o) 386 388
387
SHORT COMMUNICATIONS
We investigated the cleavage under various conditions and thus found the sites of primary attack which correspond to the isolated peptides in order of their yield (cf. Fig. I). Cleavage of synthetic substrates. A volume of 200/*1 of 0.05 M trimethylamine buffer, which had been adjusted to pH 4, 5, 6, 7, 8 or 9 by acetic acid, was mixed with 20 #1 of a solution containing 0.05/*mole of substrate in water (or in 50% ethanol) and with IO/,1 of the enzyme solution. The resulting molar enzyme to substrate ratio was always 1:20. The reaction mixture was incubated 16 h at 37 °. The cleavage was studied by paper chromatography in System S1. The sites of cleavage of substrates containing a higher number of amino acid residues were established in analogous experiments carried out on a preparative scale (I/,mole of substrate at pH 8). The isolated digestion products were characterized by amino acid analysis 11. In the pH range 7-9 the following substrates * were cleaved completely: Gly-Phe-OMe
Gly-Phe-pNa
Gly-Phe-Leu
Leu-Gly-pNa
Leu-Leu pNa
Gly-Tyr OEt
Asn-Ala P r o - L e u - G l y - N H 2
4
Boc-/%Ala-Trp-Met-Asp-Phe-N H ~ Z G l y - P r o - G l y - G l y Pro-Ala
There was no cleavage of the following substrates : Glu Asn Glu-Glu-Ala Suc-Phe-pNa
Pro-Leu-Gly-NH 2 Ac-Phe-Tyr Gly-Pro-Ala
Our results obtained concerning the digestion of synthetic peptides show that the proteinase cleaves peptide bonds at the carboxyl side of phenylalanine, leucine, tyrosine, aspartic acid, and methionine. A fact deserving interest is that the proteinase also splits the bond at the carboxyl side of glycine, if the carboxyl of the adjacent phenylalanine residues is bound by a peptidic or at least pseudopeptidic residue, not, however, if the phenylalanine carboxyl is esterified. It is likely that in some cases the substitution of the N-terminal amino acid prevents the cleavage. By contrast, the elongation of peptide P r o - L e u - G l y - N H 2 at its N-terminus rendered the peptide susceptible to cleavage. The specificity of the alkaline proteinase of A. flavus, as observed with the digestion of the B-chain of oxidized insulin, strongly resembles the specificity of the mold proteinase of A. oryzae described by SANGER et al. TM (cf. Fig. I) ; it differs, however, from the specificity of aspergillopeptidase C of A. oryzae as reported by NORDWIG AND JAHN 4. The specificity of our proteinase corresponds to the specificity of the alkaline proteinase isolated from A. oryzae by MORIHARA AND TSUZUK113. These authors, however, report for their preparation a molecular weight of 52 ooo, which is different from the value of 18 ooo-19 ooo reported by other authors3, 4 and by us 1. Our results provide additional evidence of the great similarity existing between the proteinase of A. flavus and the alkaline proteinase of A. oryzae (cf. refs. I, 2). Biochim. Biophys. Acta, 198 (197 o) 386-388
388
SHORT COMMUNICATIONS
The d a t a p r e s e n t e d a b o v e show t h a t the specificity of t h e alkaline proteinase of
A. flavus is r a t h e r b r o a d . In t h e i r s t u d y on the b i n d i n g site of papain, SCHFCHTH~ AND BERGEn 14 r e p o r t e d a seven-residue b i n d i n g site of a s p e r g i l l o p e p t i d a s e B (@ ref. 3)- F o r this reason, we a r r a n g e d all the p e p t i d e s which are cleaved b y our proteinase in such a m a n n e r t h a t the a m i n o acids e q u a l l y d i s t a n t from the site of cleavage were below each other. W h e n c o m p a r i n g the n e i g h b o r h o o d of t h e cleaved bond, we did n o t find a n y obvious regularity. The low specificity of t h e p r o t e i n a s e was in good a g r e e m e n t w i t h t h e assumed m u l t i p l i c i t y of the binding site and o b v i o u s l y plays a f u n c t i o n a l role in the proteinases of lower organisms. The results of our i n v e s t i g a t i o n of the cleavage of b o t h the t3-chain of oxidized insulin a n d also the s y n t h e t i c p e p t i d e s lead us to conclude t h a t the proteinase isolated b y us falls into the g r o u p of serine proteinases of b r o a d specificity ta, a group to which also belongs, a m o n g others, subtilisin with the same serine active center 2,~s. W e wish to acknowledge the skillful technical assistance of Mrs..]. l.uke~ovfi. W e are i n d e b t e d to Mr..J. Zbro~ek a n d Miss V. Himrovfi for amino acid analyses.
Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Science, Prague (Czechoslovakia)
.l. "I'URKOV.'~ (). MIKF.g
I J. TURKOVA,O. MIKEg, 1{. (;AN(~EVAND ,1~'I.BOUI~LiK,Biochim. Biophys. Aela, 178 (I969) ioo. 2 0 . MIKE.~, J. TURKOVA, NGUYEX BAO TOAN AND F..~ORM, Biochim. Biophys. Acta, z78 11969) 3 4 5 6 7 8 9 1o I1 I2 13 14 15 16 17
II2. A. R. SUBRAMANIAN AND (;. KALNITSKY, Biochemistry, 3 11964) i 8 6 8 . A. NORDWlG AND VV. F. JAHN, European J. Biochem., 3 (1968) 519 . F. SANGER, Proc. Chem. Soc., 11963) 76. J. TURKOV• AND O. MIKEg, Abstr. 6th Federation European Biochem. Nocs. ,]leetin/~,, Madrid 1969, No. 187 . C. H. HIRS, J. Biol. Chem., 219 11959 ) 611. B. KEIL AND H. I(EILOV,~, Collection Czech. Chem. Commtzn., 29 (t91)4) 22o(i. S. G. WALEY AND J. WATSON, Biochem. J., 55 (1953) 328. Z. PRUSfK AND B. KEIL, Collection Czech. Chem. Commun., 25 (t96o) 2o49. D. H. SPACKMAN, ~V. H . STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 119o. F. SANGER, E . O. P. THOMPSON AND R. KITAI, Biochem. ,[., 59 11955) 509. |~[. MORIHARA AND H. TSUZUKI, Arch. Biochem. Biophys., 129 (1969) 620. 1. SCHECHTER AND A. BERGER, Biochemistry, 5 (1966) 337 I. H. F. NOLLER AND S. A. BERNHARD, Biochemistry, 4 11965) 1i~8. W . R. GRAY AND B. ~. HARTLEY, Biochem. J., 89 (1963) 379. J. NOVOTN') AND t;. ];RANbZK, Chem. I_isty, 62 11968) 995.
R e c e i v e d S e p t e m b e r 29th, 1969 Biochim. Biophys. ,4cta, TOS (197 o) 386 3 , ~