[57]
PROTEINASE INHIBITORS FROM LEGUME SEEDS
697
ficient preincubation of enzyme and inhibitor should be allowed to reach inhibition equilibrium. Chapters [56]-[63] deal with the most characterized inhibitors from plant sources. As stated already by KasselP in this series, it has not been possible to express the units of inhibiting activity in a uniform manner; the assays and units are those used by the individual investigators whose isolation procedures are described. The chapter by Kassell, 8 the reviews by Liener and K a k a d e ° and by Laskowski, Jr. and Sealock, 1° and the Proceedings of the First and Second International Research Conferences on Proteinase Inhibitors 11,~ should be consulted for earlier as well as for further information. Acknowledgment The author is grateful to Dr. R. Hofstein for her most valuable assistance in preparing Chapters [56]-[63]. 8B. Kassell, this series Vol. 19 [66]. ' I . E. Liener and M. L. Kakade, in "Toxic Constituents of Plant Foodstuffs" (I. E. Liener, ed.), p. 8. Academic Press, New York, 1969. 10M. Laskowski, Jr., and R. W. Sealock, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 3, p. 375. Academic Press, New York, 1971. 11Proteinase Inhibitors Proc. First Int. Res. Con]. 1st, Munich, 1970, de Gruyter, Berlin, 1971. 12Proteinase Inhibitors, Proc., Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973. Springer-Verlag, Berlin and New York, 1974.
[57] Proteinase
Inhibitors
from
Legume Seeds
B y YEHUDITH BIRK
The presence of proteinlike trypsin inhibitors in legume seeds is by now a well established fact. Different legume seeds contain one or more inhibitors. Most of them have a molecular weight of about 8000 with a high concentration of cystine ( ~ 2 0 % ) and no free SH groups. The abundance of S - - S bonds accounts for the considerable resistance to overall denaturation and to proteolytic digestion. Establishment of the amino acid sequence of the B o w m a n - B i r k soybean inhibitor 1 and of the lima bean inhibitor I V 2 shows the existence of two homologous regions in these proteins. The region located in the first half of the molecule S. Odani and T. Ikenaka, J. Biochem. (Tokyo) 71,839 (1972). : F. C. Stevens, S. Wuerz, and J. Krahn, Proteinase Inhibitors, Proc. Int. Res. Con/., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 344. Springer-Verlag, Berlin and New York, 1974.
698
BB
NATURALLY
OCCURRING PROTEASE INHIBITORS
[57]
LB
Asp-Asp-Glu-Ser-Ser-Lys-Pro-CyslO Ser-Gly-His-His-Glu-His-Ser-Thr-Asp-Glx-Pro-Ser -Glx-Ser-Ser-Lys-Pro-Cys-
BB
10 20 Cys-Asp-Gln-Cys-Aia -Cys~Lys~-~AsnfPro-Pro-Gln -Cys-Arg-Cys-Ser-Asp-
GB
(Val, Cys)lThrtAla~SertIle tPro-p3r°-Gln (Cys,Ile, Cys, Thr, Asx,
LB
Cys-A2s°n-His-Cys[AlaT-Cys T ~ r LysS~er Ile ~Pro-p3r°-Gln -Cys-Arg-Cys-Thr-AspSet ~euJ
BB
Met~A
GB
Val) IArg-Leut 4A4~x• Ser-Cys-His-Ser-Ala-Cys-Lys-Ser-Cyst Met 5° ~Cyst Thr-Arg~Sert
LB
Leu-~Arg-Leu-~AspSer-Cys-His-Ser-Ala-Cys-Lys-Ser-Cys~I510C~ys Thr-LeuS~er Phe
3° Ser-Cys-His-Ser-Ala-Cys-Lys-Ser-Cys~Ile 40 ~ - ~ h l a -Leu~-~-
LB
50 60 Tyr ~Ala-Gln~Phe-Cys-Val-Asp-Ile -Thr-Asp-Phe~Glu-Pro~Cysi t i i 8° ] i 70 ] Met]-Pro-~Gly-Lys~CysfArg-Cys-Leu-Asx-Thr-Thr-Asx-Tyr~-Cys-Tyr]-Lys-Serj-CysI I I L 6° I L 7° / Ile PL~-Ala-Gln C~_~ Val (Cys,Thr, Asx)Ile-Asx-Asp-PheJCys-Tyr~-Glu-Pro[-Cys-
BB
7O LysiPro-Ser-Glu . . . . . Asp-Asp-Lys-Glu-Asn
GB
LystSer -Asx-Ser-Gly-Glx-Asx-Asx
LB
LyslSer-Ser-His-Ser-Asp-Asp-ASsp-Asn-Asn-Asn
BB GB
contains the trypsin inhibitory site, and the one located in the second half of the molecule contains the chymotrypsin inhibitory site. The striking similarity between the Bowman-Birk inhibitor and the lima bean inhibitor is also expressed in their antigenicity, since specific antibodies to the former cross-react with the latter and vice versa2 A very high degree of homology exists between these two inhibitors and garden bean inhibitor II. 4 This can be seen from the accompanying comparison (where BB, GB, and LB stand for the Bowman-Birk soybean inhibitor, garden bean inhibitor II, and lima bean inhibitor IV, respectively). However, in the
3y. Birk, Proteinase lnhibitors, Proc. Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 355. Springer-Verlag, Berlin and New York, 1974. 4K. A. Wilson and M. Laskowski, Sr., Proteinase Inhibitors Proc. Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 286. Springer-Verlag, Berlin and New York, 1974.
[57[
PROTEINASE
INHIBITORS
FROM
LEGUME
SEEDS
L ¢p
© o
.< 0 0
a~
o q~
Z ~
Z
Z
¢q Z
q~
< © Z
N
N IN
e~ m
N
N
0
t-* ~
699
700
NATURALLY OCCURRING PROTEASE INHIBITORS
[58]
garden bean inhibitor II the trypsin reactive site is located in the second half of the molecule, at a position homologous to the chymotrypsinreactive sites of the Bowman-Birk and lima bean inhibitors. Comparison of the reactive sites of the Bowman-Birk soybean inhibitor (AA) with those of the groundnut inhibitor (GI) and the chick pea inhibitor (CI) 3 indicate that, although all the three are double-headed inhibitors, the reactive sites in CI are overlapping and the trypsin-reactive site in GI is not the trypsin-inhibitory site (see the table).
[58] T r y p s i n a n d C h y m o t r y p s i n I n h i b i t o r s f r o m S o y b e a n s B y YEHUDITH BIRK
The two prevalent trypsin inhibitors in soybeans are the Kunitz soybean inhibitor (SBTI) and the Bowman-Birk inhibitor (inhibitor AA). They differ markedly from each other in size, amino acid composition, structure, and properties. A detailed description of the preparation of these two inhibitors, as well as their physical and kinetic properties and specificity are outlined by Kassell in this series, Vol. 19 [66c]. Only certain selected features of the inhibitors, such as their amino acid sequence, their susceptibility to modifications, and their biological activities, will be mentioned here.
Kunitz Soybean Trypsin Inhibitor (SBTI)
Properties K i n e t i c Properties. 1-3 The kinetics of interaction of the inhibitor with bovine fl-trypsin have been studied extensively by Laskowski, and
1M. Laskowski, Jr., R. W. Duran, W. R. Finkenstadt, S. Herbert, H. F. Hixson, D. Kowalski, J. A. Luthy, J. A. Mattis, R. E. McKee, and C. W. Niekamp, Proteinase Inhibitors, Proc. Int. Res. Con]., 1st, Munich, 1970, p. 117. de Gruyter, Berlin, 1971. 2j. A. Luthy, M. Praissman, W. R. Finkenstadt, and M. Laskowski, Jr., J. Biol. Chem. 248, 1760 (1973). 'W. R. Finkenstadt, M. A. Hamid, J. A. Mattis, J. Schrode, R. W. Sealock, D. Wang, and M. Laskowski, Jr., Proteinase Inhibitors, Proc. Int. Res. Conf., 2nd (Bayer Syrup. V), Grosse Ledder, 1973, p. 389. Springer-Verlag, Berlin and New York, 1974.
PROTEINASE INHIBITORS FROM LEGUME SEEDS
697
ficient preincubation of enzyme and inhibitor should be allowed to reach inhibition equilibrium. Chapters [56]-[63] deal with the most characterized inhibitors from plant sources. As stated already by KasselP in this series, it has not been possible to express the units of inhibiting activity in a uniform manner; the assays and units are those used by the individual investigators whose isolation procedures are described. The chapter by Kassell, 8 the reviews by Liener and K a k a d e ° and by Laskowski, Jr. and Sealock, 1° and the Proceedings of the First and Second International Research Conferences on Proteinase Inhibitors 11,~ should be consulted for earlier as well as for further information. Acknowledgment The author is grateful to Dr. R. Hofstein for her most valuable assistance in preparing Chapters [56]-[63]. 8B. Kassell, this series Vol. 19 [66]. ' I . E. Liener and M. L. Kakade, in "Toxic Constituents of Plant Foodstuffs" (I. E. Liener, ed.), p. 8. Academic Press, New York, 1969. 10M. Laskowski, Jr., and R. W. Sealock, in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 3, p. 375. Academic Press, New York, 1971. 11Proteinase Inhibitors Proc. First Int. Res. Con]. 1st, Munich, 1970, de Gruyter, Berlin, 1971. 12Proteinase Inhibitors, Proc., Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973. Springer-Verlag, Berlin and New York, 1974.
[57] Proteinase
Inhibitors
from
Legume Seeds
B y YEHUDITH BIRK
The presence of proteinlike trypsin inhibitors in legume seeds is by now a well established fact. Different legume seeds contain one or more inhibitors. Most of them have a molecular weight of about 8000 with a high concentration of cystine ( ~ 2 0 % ) and no free SH groups. The abundance of S - - S bonds accounts for the considerable resistance to overall denaturation and to proteolytic digestion. Establishment of the amino acid sequence of the B o w m a n - B i r k soybean inhibitor 1 and of the lima bean inhibitor I V 2 shows the existence of two homologous regions in these proteins. The region located in the first half of the molecule S. Odani and T. Ikenaka, J. Biochem. (Tokyo) 71,839 (1972). : F. C. Stevens, S. Wuerz, and J. Krahn, Proteinase Inhibitors, Proc. Int. Res. Con/., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 344. Springer-Verlag, Berlin and New York, 1974.
698
BB
NATURALLY
OCCURRING PROTEASE INHIBITORS
[57]
LB
Asp-Asp-Glu-Ser-Ser-Lys-Pro-CyslO Ser-Gly-His-His-Glu-His-Ser-Thr-Asp-Glx-Pro-Ser -Glx-Ser-Ser-Lys-Pro-Cys-
BB
10 20 Cys-Asp-Gln-Cys-Aia -Cys~Lys~-~AsnfPro-Pro-Gln -Cys-Arg-Cys-Ser-Asp-
GB
(Val, Cys)lThrtAla~SertIle tPro-p3r°-Gln (Cys,Ile, Cys, Thr, Asx,
LB
Cys-A2s°n-His-Cys[AlaT-Cys T ~ r LysS~er Ile ~Pro-p3r°-Gln -Cys-Arg-Cys-Thr-AspSet ~euJ
BB
Met~A
GB
Val) IArg-Leut 4A4~x• Ser-Cys-His-Ser-Ala-Cys-Lys-Ser-Cyst Met 5° ~Cyst Thr-Arg~Sert
LB
Leu-~Arg-Leu-~AspSer-Cys-His-Ser-Ala-Cys-Lys-Ser-Cys~I510C~ys Thr-LeuS~er Phe
3° Ser-Cys-His-Ser-Ala-Cys-Lys-Ser-Cys~Ile 40 ~ - ~ h l a -Leu~-~-
LB
50 60 Tyr ~Ala-Gln~Phe-Cys-Val-Asp-Ile -Thr-Asp-Phe~Glu-Pro~Cysi t i i 8° ] i 70 ] Met]-Pro-~Gly-Lys~CysfArg-Cys-Leu-Asx-Thr-Thr-Asx-Tyr~-Cys-Tyr]-Lys-Serj-CysI I I L 6° I L 7° / Ile PL~-Ala-Gln C~_~ Val (Cys,Thr, Asx)Ile-Asx-Asp-PheJCys-Tyr~-Glu-Pro[-Cys-
BB
7O LysiPro-Ser-Glu . . . . . Asp-Asp-Lys-Glu-Asn
GB
LystSer -Asx-Ser-Gly-Glx-Asx-Asx
LB
LyslSer-Ser-His-Ser-Asp-Asp-ASsp-Asn-Asn-Asn
BB GB
contains the trypsin inhibitory site, and the one located in the second half of the molecule contains the chymotrypsin inhibitory site. The striking similarity between the Bowman-Birk inhibitor and the lima bean inhibitor is also expressed in their antigenicity, since specific antibodies to the former cross-react with the latter and vice versa2 A very high degree of homology exists between these two inhibitors and garden bean inhibitor II. 4 This can be seen from the accompanying comparison (where BB, GB, and LB stand for the Bowman-Birk soybean inhibitor, garden bean inhibitor II, and lima bean inhibitor IV, respectively). However, in the
3y. Birk, Proteinase lnhibitors, Proc. Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 355. Springer-Verlag, Berlin and New York, 1974. 4K. A. Wilson and M. Laskowski, Sr., Proteinase Inhibitors Proc. Int. Res. Con]., 2nd (Bayer Symp. V), Grosse Ledder, 1973, p. 286. Springer-Verlag, Berlin and New York, 1974.
[57[
PROTEINASE
INHIBITORS
FROM
LEGUME
SEEDS
L ¢p
© o
.< 0 0
a~
o q~
Z ~
Z
Z
¢q Z
q~
< © Z
N
N IN
e~ m
N
N
0
t-* ~
699
700
NATURALLY OCCURRING PROTEASE INHIBITORS
[58]
garden bean inhibitor II the trypsin reactive site is located in the second half of the molecule, at a position homologous to the chymotrypsinreactive sites of the Bowman-Birk and lima bean inhibitors. Comparison of the reactive sites of the Bowman-Birk soybean inhibitor (AA) with those of the groundnut inhibitor (GI) and the chick pea inhibitor (CI) 3 indicate that, although all the three are double-headed inhibitors, the reactive sites in CI are overlapping and the trypsin-reactive site in GI is not the trypsin-inhibitory site (see the table).
[58] T r y p s i n a n d C h y m o t r y p s i n I n h i b i t o r s f r o m S o y b e a n s B y YEHUDITH BIRK
The two prevalent trypsin inhibitors in soybeans are the Kunitz soybean inhibitor (SBTI) and the Bowman-Birk inhibitor (inhibitor AA). They differ markedly from each other in size, amino acid composition, structure, and properties. A detailed description of the preparation of these two inhibitors, as well as their physical and kinetic properties and specificity are outlined by Kassell in this series, Vol. 19 [66c]. Only certain selected features of the inhibitors, such as their amino acid sequence, their susceptibility to modifications, and their biological activities, will be mentioned here.
Kunitz Soybean Trypsin Inhibitor (SBTI)
Properties K i n e t i c Properties. 1-3 The kinetics of interaction of the inhibitor with bovine fl-trypsin have been studied extensively by Laskowski, and
1M. Laskowski, Jr., R. W. Duran, W. R. Finkenstadt, S. Herbert, H. F. Hixson, D. Kowalski, J. A. Luthy, J. A. Mattis, R. E. McKee, and C. W. Niekamp, Proteinase Inhibitors, Proc. Int. Res. Con]., 1st, Munich, 1970, p. 117. de Gruyter, Berlin, 1971. 2j. A. Luthy, M. Praissman, W. R. Finkenstadt, and M. Laskowski, Jr., J. Biol. Chem. 248, 1760 (1973). 'W. R. Finkenstadt, M. A. Hamid, J. A. Mattis, J. Schrode, R. W. Sealock, D. Wang, and M. Laskowski, Jr., Proteinase Inhibitors, Proc. Int. Res. Conf., 2nd (Bayer Syrup. V), Grosse Ledder, 1973, p. 389. Springer-Verlag, Berlin and New York, 1974.
