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[58] Carboxypeptidase inhibitor from potatoes
778
INHIBITORS OF VARIOUS SPECIFICITIES
[58]
both male and female chickens, so the synthesis of the protein is not confined to the female reproductive tract. Skin and other tissues of all the mammals examined so far contain inhibitors with properties similar to egg white cystatin, and it seems possible that these are members of a single homologous family of cysteineproteinase inhibitors. '~-'9 Common characteristics of the cystatin-type inhibitors are Mr close to 13,000, inhibition of papain and cathepsin B (with interaction even when the catalytic site is blocked), inhibition of dipeptidyl peptidase I but scarcely of bromelain, pI 4.5-6.5, and stability to heat and alkali. Amino acid sequence data will be required to confirm or deny the possibility of homology, however. 1~ K. Udaka and H. Hayashi, Biochim. Biophys. Acta 104, 600 (1965). ~7 M. J~.rvinen, Acta Chem. Stand.. Set'. B 30, 933 (1976). '~ M. Jhrvinen, J. Invest. Dermatol. 72, 114 (1978). ,9 T. Hibino, K. Fukuyama, and W. L. Epstein, Biochim. Biophys. Acta 632, 214 (1980).
[58]
Carboxypeptidase Inhibitor from Potatoes By G. MICHAEL HASS and C. A. RYAN
Although proteinaceous inhibitors of the serine proteases are widely distributed in nature, polypeptides that specifically inhibit the pancreatic carboxypeptidases have only been found in potatoes,' tomatoes," and roundworms. 3 The inhibitors from potatoes and tomatoes are highly homologous/and are believed to function both as storage proteins and as a defense mechanism by arresting digestive proteolysis of invading pests. The availability of relatively large amounts of the carboxypeptidase inhibitor from potatoes has facilitated extensive investigations of its structure and mechanism of action. In addition, affinity chromatography utilizing immobilized inhibitor has proved to be an effective and economical means of purifying a variety of metallocarboxypeptidases that are target enzymes for the inhibitor. Assay Procedure The carboxypeptidase inhibitor (CPI) is usually assayed by measuring the inhibition of carboxypeptidase A activity. The substrate most corn'
J. M. Rancour and C. A. Ryan, Arch. Biochem. Biophys. 125, 380 (1968). G. M. Hass and C. A. Ryan, Phytochemistry 19, 1329 (1980). 3 G. A. Homandberg and R. J. Peanasky, J. Biol. Chem. 251, 2226 (1976). 4 G. M. Hass and M. A. Hermodson, Plant Physiol. 65, 33 (1980).
METHODS IN ENZYMOLOGY,VOL. 80
Copyright © 1981 by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181980-9
[58]
POTATO CARBOXYPEPTIDASE INHIBITOR
779
monly employed is hippuryl-L-phenylalanine,5"~ although chloroacetyl-Ltyrosine 7 and hippuryl-DL-phenyllactate are also commonly used. A radial diffusion immunoassay for screening CPI levels in large numbers of samples has also been employed~; however, a source of anti-carboxypeptidase inhibitor antibodies is required for this procedure. Reagents
Substrate: Hippuryl-L-phenylalanine (1 mM) in 0.5 M NaCI-20 mM Tris-HC1 (pH 7.5) Enzyme: Carboxypeptidase A (Worthington or Sigma) is dissolved in 5 M NaCI-0.05 M Tris-HC1 (pH 7.5) at 4°C, and diluted with assay buffer in a concentration of approximately 0.1 mg/ml. The protein concentration can be estimated by absorbance using • E2~0nm 0 l°f¢~ = 1.88. Inhibitor: The stock inhibitor solution should be diluted with buffer such that an aliquot of 20-50 ~1 produces 25-70% inhibition. Procedure Substrate (3ml) is equilibrated at 25°C in a recording spectrophotometer. Controls are performed by adding enzyme alone (20 ~1) and monitoring the increase in absorbance at 254 nm. The inhibitor is detected as a decrease in the rate of change in absorbance. Since equilibration between enzyme, inhibitor, and substrate is rapid, there is no need for preincubation.
Data Analysis The amount of inhibitor present in the aliquot taken for assay is given by the following: CPI (tzmol) - % Inhibition CPA (/zg) 100 × 34,500/zg/tzmol where CPA is the amount of carboxypeptidase A present in the assay solution. This expression is valid for approximately 0-70% inhibition using hippuryl-L-phenylalanine as substrate and for 0-95% inhibition with chloroacetyl-L--tyrosine. The low Km for hippuryl-oL-phenyllactate and low enzyme concentration used in the assays render impractical the titration of enzymatic activity with this substrate. J. E. Folk and E. W. Schirmer, J. Biol. Chem. 238, 3884 (1963). ~;C. A. Ryan, G. M. Hass, and R. W. Kuhn, J. Biol. Chem. 249, 5495 (1974). G. M. Hass, H. Ako, D. G. Grahn, and H. Neurath, Biochemistry 15, 93 (1976). C. A. Ryan, T. Kuo, G. Pearce, and R. Kunkel, Am. Potato J. 53, 443 (1976).
780
INHIBITORSOF VARIOUSSPEC1F1CITIES I
[58]
I
12 Vo IO
• 0 ~ z
0.8 INHIBITOR I
0.6
A
m
m 04
02
i
0
2O
3 TUBE NUMBER (40 ml/tube)
FIG. 1. Chromatography of crude inhibitor I on Sephadex G-75. (See Ryan eta/. ~ for details.)
Purification Method
The purification of the principal carboxypeptidase isoinhibitor has been previously described in this series2 Briefly, this procedure involves homogenization of Russet Burbank potato tubers in H20 containing sodium dithionite, acidification to pH 3.0, ammonium sulfate fractionation (0-70% saturation), heat treatment (80c~ for 5 rain), exhaustive dialysis against H20, and lyophilization. This partially purified inhibitor (crude inhibitor I) is resolved into several zones by gel filtration on Sephadex G-75 (Fig. 1). The first inhibitor to elute from Sephadex G-75 is inhibitor I (MW -40,000'°), the second is inhibitor II (MW -20,00011), and in a "polypeptide p e a k " (fractions 77-104), there are several low-molecular-weight inhibitors of trypsin and c h y m o t r y p s i n and the carboxyY. Birk, this series, Vol. 46, p. 736. 11,j. C. Melville and C. A. Ryan, J. Bh)l. Chem. 249, 5495 (1972). ~ J. Bryant, T. R. Greene, T. Gurusaddaiah, and C. A. Ryan, Bh)chemistry 15, 3418 (1976).
[58]
POTATO CARBOXYPEPTIDASE INHIBITOR
781
peptidase isoinhibitors. Ion exchange is utilized to purify the inhibitor further. In earlier reports phosphocellulose was employed~'9; however, carboxymethyl cellulose appears to give better resolution of the many proteinase inhibitors present in the polypeptide fraction and is the preferred resin. After lyophilization, polypeptide material derived from approximately 14 kg of potatoes is dissolved in 5 mM sodium citrate (pH 4.3) and chromatographed on a column (2.5 × 40 cm) of carboxymethyl cellulose (Whatman CM-52) (Fig. 2). Following elution of the breakthrough, the column is developed with a linear gradient (2 liters total) of NaC1 (0-0.5 M) in equilibration buffer. Three zones exhibiting carboxypeptidase-inhibitory activity (Fig. 2) are observed and the three pools are lyophilized and desalted by gel filtration on Sephadex G-25. Isoinhibitor II, the predominant species, is apparently homogeneous at this stage, whereas additional chromatography steps are required to purify completely the minor isoinhibitors, CPI-I and CPI-III. v-' Yieht
The amount of carboxypeptidase inhibitor differs considerably among varieties of potatoes, ~ and it is recommended that a sampling of tubers be homogenized and assayed before engaging in a large-scale preparation. Russet Burbank potatoes grown in the states of Idaho and Washington typically yield 200 mg CPI-II, 25 mg CPI-I, and 25 mg CPI-III per 50 kg (fresh weight). Purity
The homogeneity of CPI preparations can be easily assessed by polyacrylamide-gel electrophoresis ~ or by isoelectric focusing.'2 Alternatively, the absence of leucinC in CPI is a convenient monitor that allows detection of most protein contaminants after acid hydrolysis and amino acid analysis.
Characterization Physical and Chemical Properties
CPI-I1 is an unusually stable protein easily withstanding temperatures up to 90°C and extremes of pH (pH 2-11.5) for extended time periods. Even in the tuber it is extraordinarily stable during cooking by several ~z G. M. Hass, J. E. Derr, D. J. Makus, and C. A. Ryan, Plant Phv.siol. 64, 1022 (1979).
782
INHIBITORS
OF VARIOUS SPECIFICITIES
[58]
IA[ 'IODN 0 I
0 I
"\
0 I
O CO
\ \
O tO
\ \
\ \
o
d::
-I O oa
o°
'~
E
Z
.o
.o
~
!
o w u 0~3~
io ~ o u o q J o s q v
.=-=_
[58]
783
POTATO CARBOXYPEPTIDASE INHIBITOR TABLE I CHARACTERISTICS OF CPIs FROM POTATO TUBERS Property"
CPI-I
CPI-II
CPI-III
Molecular weight Isoelectric point N-terminus K , , , , . (CPA) Ki,app. (CPB)
4089, 4219 ~' 4.6
4204, 4333 b 5.8
4075 6.5 His 1.6-3.3 n,f/ 1.3-3.3 n ~ /
" Data are taken from Hass et al. ,z b Data given for 38- and 39-residue species (see Hass et al. '~ and Fig. 2).
methods? 3 The properties of all three of the isoinhibitor species are given in Table I. Molecular weights (-3000) estimated by gel filtration are somewhat lower than the values determined by amino acid sequence analysis, presumably because the inhibitors are rather hydrophobic, and thus are retarded during chromatography. 6"12 The amino acid compositions of the carboxypeptidase isoinhibitors are given in Table II. 12 The isoinhibitors contain neither methionine nor leucine, and CPI-I lacks arginine as well. The extinction coefficient E°8}~rn = 3.0, determined by titration of stock solutions against carboxypeptidase A, is in good agreement with that predicted from the content of aromatic amino acids.
Amino Acid Sequence The principal form of the inhibitor (CPI-II) is a nearly equimolar mixture of the 38- and 39-amino acid residue peptides indicated in Fig. 3.14 This amino acid sequence, determined primarily by convential procedures, 14 has been confirmed by GC-MS analysis of suitably derivatized peptide fragments? ~ The two isoinhibitors may be separated by ionexchange chromatography, although this is not usually necessary. CPI-I differs from CPI-II at two positions (Sera0 --~ Ala and Arg32 --~ Gly). CPI-III is identical to CPI-II except that it lacks the amino-terminal pyrrolidone carboxylic acid and the adjacent glutamine residue. The six residues of half-cystine of CPI are found as 3 disulfide bonds (Cyss-Cys24, C y s 1 2 - C y s 2 7 , and C y s l s - C y s 3 4 ) . 16 ,:5 D. Y. H u a n g , B. G. Swanson, and C. A. Ryan, J. F o o d Sci. 46, 287 (1981). ,4 G. M. Hass, H. Nau, K. B i e m a n n , D. T. Grahn, L. H. Ericsson, and H. Neurath, B i o c h e m i s t r y 14, 1334 (1975). ~:' H. N a u and K. Biemann, B i o c h e m . B i o p h y s . R e s . C o m m u n . 73, 175 (1976). "~ T. R. Leary, D. T. Grahn, H. Neurath, and G. M. Hass, B i o c h e m i s t o , 18, 2252 (1979).
784
INHIBITORS OF VARIOUS SPECIFICITIES
[58]
TABLE II AMINO ACID COMPOSIrlON OF CPls Calculated no. of amino acid residues per molecule CPI-I
CPI-II
CPI-III
Amino acid
Average"
Integral
Average"
Integral
Average"
Integral
Asp Thr Ser Glu Pro Gly Ala Val Ile Tyr Phe His Lys Arg Trp ½Cys
5.00 1.91 1.04 2.66 3.25 3.74 4.83 1.27 0.85 1.08 1.13 2.01 1.89 0.01 1.69 5.75
5 2 1 2-3 3 4 5 1 1 1 1 2 2 0 1-2 6
4.96 1.95 1.98 2.42 2.94 2.96 4.00 0.98 0.90 0.99 1.03 2.15 2.13 1.05 1.3 5.8
5 2 2 2-3 3 3 4 1 1 1 l 2 2 1 1-2 6
5.00 1.92 1.75 1.15 3.28 3.02 4.18 0.95 0.95 1.1! 1.24 2.14 2.20 1.09 1.23 5.85
5 2 2 1 3 3 4 1 1 1 1 2 2 1 1-2 6
" Taken from Hass
Potato 1
Tomato 1
e t a l . v-,
His Ala His Ala
Ile ....
Asn
Lys Pro Cys
Lys
His
Gln Tyr
Val
His |Lys Pro Cys
Ser
Gln
[
!
Potato 16 ~Asp Asp Cys Ser Gly
Ala Trp IPhe Cys Gln Ala Cys Trpl Asn Ser
! Tomato 16 ~Asp Asp Cys Ser Gly
V-q
Gly Thr
IPhe
I
Cys Gln Ala Cys Trpl Arg Phe
Potato 31 IAlal Arg
Thr Cys Gly Pro Tyr Val
Tomato 31 ~
Thr Cys Gly Pro Tyr Val oH I
Gly
Gly OH
FIG. 3. A comparison of the amino acid sequences of the carboxypeptidase inhibitors from potato tubers and tomato fruit. Both 38- and 39-residue forms of CPI-II from potatoes ~2 are shown. The residues enclosed in boxes are identical in both sequences. Sequence data are taken from Hass e t a l . 4.,~
[58]
POTATO CARBOXYPEPTIDASE INHIBITOR
30
785
I0
2O
J 0 Zn
279 ~ °I
127 71
0 En
2790,.
71
FKi. 4. Stereoview of the carboxypeptidase inhibitor in association with carboxypeptidase A as determined by X-ray diffraction at 2.5 A, resolution. ~
The amino acid sequence of CPI demonstrates that it is homologous to the carboxypeptidase inhibitor from tomato fruit (Fig. 3), and suggests that it may be homologous to a polypeptide chymotrypsin inhibitor from potatoesJ 7 as well as to several toxic peptides including viscotoxin A2 from the European mistletoe and/J-purothinine from wheatJ 8 although these latter comparisons are highly speculative and are based primarily on the half-cystine residues. Three-Dimensional Structure
The structure of CPI-II has been deduced by X-ray diffraction analysis at 2.5 A resolution of the inhibitor-carboxypeptidase A complex? 9 CPI-II has neither helical structures nor/J-pleated sheets, as is seen in Fig. 4. X - R a y Diffraction Analysis
The interaction of the carboxyl-terminal region of the inhibitor from potatoes with carboxypeptidases has been clearly indicated by X-ray crystallographic analysis of the enzyme-inhibitor complex at 2.5 A resolution TM (Fig. 4). That report demonstrates that the inhibitor binds like an extended substrate. The most unusual features of the inhibitor-enzyme ~7 G. M. Hass, R. Venkatakrishnan, and C. A. Ryan, Proc. Natl. Acad. Sci. U.S.A. 73, 1941
(1976). ~ D. J. Strydom,J. Mol. Evol. 9, 349 (1977). ~ D. C. Rees and W. N. Lipscomb,Pro('. Natl. Acad. Sci. U.S.A. 77, 4633 (1980).
786
INHtBITORS OF VARIOUS SPECIFICITIES
[58]
interaction are that the Val38-Gly39 peptide bond is cleaved in the complex, and that GIy39 appears to be trapped in the binding pocket of the enzyme. 19 The comparison of the amino acid sequences of the inhibitors from tomatoes and potatoes (Fig. 3) is of particular interest in this respect. The amino acid residue that would correspond to Gly39 of the inhibitor from potatoes is not found in the inhibitor from tomatoes. This observation has led to recent studies demonstrating that carboxypeptidase A rapidly removes Gly39 from the potato inhibitor and that this residue is not required for enzyme-inhibitor interaction. 26 The X-ray diffraction analysis of the enzyme-inhibitor complex 19 indicates that residues 37-39 of the inhibitor interact extensively with the enzyme active site, an observation consistent with the complete homology in this region between the inhibitor from potatoes and from tomatoes (Fig. 3). Additional enzyme-inhibitor contacts involve the rings of Trp28, Phe22, and, to a lesser extent, HiSl~. Of these residues the only replacement observed in His15 ~ Gln (Fig. 3). The final enzyme-inhibitor contact involves the backbone of Trp~s-Ser30 of the inhibitor and Ile24T, Tyr24s, and Lys~9 of the enzyme. Although Trp28 is conserved, nonconservative replacements are found at residues 29 and 30. Apparently, these replacements do not significantly affect the conformation of the polypeptide backbone in this region. 1~ The structure is extremely compact with the disulfide bond joining Cys,s and C y s 3 4 passing through a closed loop that involves the other disulfide bridges. The structure indicated that the Val~8 and Gly39 bond was broken but that the GIy39was trapped in the specificity pocket of the enzyme (see later). Unfortunately, a structure based on X-ray diffraction of crystals of free inhibitor is not yet available. Thus the possibility that conformation changes occur in the inhibitor other than the breaking of the Va138-Gly39bond on binding enzyme has not been investigated.
Inhibition Spectrum CPI was first detected as an impurity in crystals of inhibitor I by its ability to inhibit porcine carboxypeptidase B. ''2° The inhibitor was purified and demonstrated to affect bovine carboxypeptidase A. 6 It has now been tested against a variety of carboxypeptidases from animal, plant, fungal, and bacterial sources. 21 The association between each enzyme and CPI was monitored either by inhibition of enzymatic activity ([CPI] approx. 1-10 nM) or by the binding of enzyme to CPI-Sepharose. Enzymes that participate in the degradation of dietary protein were par~ C. A. Ryan, Biochemist13' 5, 1592 (1966). .2, G. M. Hass, S. P. Ager, and D. J. Makus, Fed. Proc., Fed. Am. Soc. Exp. Biol. 39, 1689 (1980).
[58]
787
POTATO CARBOXYPEPTIDASE INHIBITOR T A B L E III BINDING OF CP1 TO DIGESTIVE TRACT CARBOXYPEPTIDASES AND TO THEIR ZYMOGENS a Procedure
Enzyme
Inhibition of e n z y m a t i c activi t y
Bovine CPDase A Bovine CPDase B Porcine CPDase A Porcine CPDase B Shrimp CPDase A Shrimp CPDase B Lungfish CPDase B Crayfish CPDase Cab bage looper C P D a s e L i m p e t CPDase Bovine Pro-CPDase A Bovine Pro-CPDase B Porcine Pro-CPDase A Porcine Pro-CPDase B Lungfish Pro-CPDase B
+ + + + + + + + + + NA ~ NA NA NA
Binding to CPI-Sepharose + + + + ND b ND ND + ND +
-
" Data are taken from Hass et al. ~ ~' ND, not determined. " NA, not applicable.
tially purified from animal species as diverse as the cow and the limpet, and all were potently affected by the inhibitor (Table III). H o w e v e r , several zymogens of the enzymes in this group were tested and shown not to bind immobilized inhibitor (Table III). With the exception of an enzyme from mast cells and a novel carboxypeptidase A-like enzyme from bovine placenta, all animal carboxypeptidases that were not of digestive tract origin were not affected by the inhibitor (Table IV). The inhibitor had no effect on the enzymatic activities of all plant and most microbial carboxypeptidases (Table V) tested. H o w e v e r , a strong association between the inhibitor and Streptomyces griseus carboxypeptidase has been noted, 22 and a low affinity (K~ - 1 0 -5 M) for a carboxypeptidase G~ from an Achtetobacter was found as well. This inhibition spectrum not only elucidates possible evolutionary relationships among the carboxypeptidases, but also identifies enzymes whose purification might be facilitated by using immobilized inhibitor as described next. ~ L. Gage-White, B. E. Hunt, G. M. Hass, and W. M. Awad, Jr., Fed. Proc.. Fed. Am. Soc. Exp. Biol. 36, 892 (1977).
788
INHIBITORS OF VARIOUS SPECIFICITIES
[58]
T A B L E IV BINDING OF CPl ~o INTRACELLULAR AND REGULATORY CARBOXYPEPTIDASES"
Procedure
Enzyme
Inhibition of
Binding of
enzymatic activity
CPI-Sepharose
Mast cell CPDase (rat) Placenta CPDase (bovine) Cathepsin A (bovine spleen) Catheptic CPDase B (bovine spleen) CPDase P (bovine kidney) CPDase N (human, bovine, porcine serum) CPDase (chicken muscle) Kininase II
+
N D t, ND ND ND ND
" Data are taken from Hass eta/.'-" ~' ND, not determined.
Mechanism of Action The inhibition of bovine carboxypeptidase A and porcine carboxypeptidase B is competitive with K~ values of approximately 5 and 50 riM, respectively. 6 Because CPI binds much tighter than model substrates to the carboxypeptidases and because CPI represents a new class of inhibitors, much effort has been directed at probing the mode of binding of this inhibitor. TABLE V BINDING OF CPI l o MICROBIAL CARBOXYPEPTIDASES 't
Procedure
Inhibition of
Binding to
Enzyme
e n z y m a t i c activity
CPI-Sepharose
CPDase GI (P. ~t,tzeri) CPDase G1 (Acinetobacter sp.) CPDase G1 (Flal'oba('terium sp.) S. g,'ise,s CPDase Yeast protease ~ Yeast protease Y P. omnivorum CPDase S. ,~clerotiorllm CPDase
+ + +
" Data are taken from Hass et al.'-" t, ND, not determined.
-
ND ~ + ND ND ND ND
[58]
1
Pro Ile Cys I Asn l
......
16
31
789
POTATO CARBOXYPEPTIDASE INHIBITOR
Cys Ser Gly Ala
Ala
Pro Cys
Thr
%__,
Phe Cys Gln Ala Cys
I Asn Ser I f L .... .J
C" . . . . I I Thr Cys Gly Pro Tyr Val 11Gly OH I I. . . . .
I
FIG. 5. A m i n o acid residues of the carboxypeptidase inhibitor from potatoes lhat do not contribute significantly to the free energy of association of the e n z y m e - i n h i b i t o r complex are enclosed in boxes. Evidence is based on a comparison with the sequence o f the inhibitor from tomatoes (___)4 and chemical modification studies ( ).7
Chemical Modification Studies The interaction in solution of CPI with target enzymes has been investigated by modifying either the inhibitor or the enzyme. First, several chemical derivatives of the inhibitor were prepared and their Ki values with respect to carboxypeptidases A and B determined to evaluate the effect of each modification on enzyme-inhibitor interaction£ Residues I-5 could be removed, the side-chain carboxylate groups modified, lysines at positions 10 and 13 acetylated, or the arginine at position 31 reacted with phenylglyoxal without affecting the strength of binding to either carboxypeptidase. Tyrosine-37 and the alpha-carboxylate of glycine-39, however, appear to be involved in contact with target enzymes (Fig. 5). In all cases, parallel effects of a given modification on the binding to both carboxypeptidases were observed, indicating that similar or identical contacts between enzyme and inhibitor were utilized in both systems.7 A second study of CPI binding to derivatives of carboxypeptidase A revealed that the binding of the inhibitor to arsanilazocarboxypeptidase A produced spectral changes similar to those observed when typical competitive inhibitors bind to this enzyme derivative. 23 Enzymatic activity was not required for association with inhibitor, since apocarboxypeptidase bound inhibitor. This situation was analogous to the tight association between anhydrotrypsin and anhydrochymotrypsin and their respective proteinase inhibitors.24 On the other hand, blockage of the binding pocket of carboxypeptidase A by reaction with N-bromoacetyl-N-methyl-L-phenylalanine had little effect on the free energy of association with inhibitor, whereas a similar modification of trypsin or chymotrypsin greatly weakens their interactions with proteinase inhibitors. ~ 2:~ H. Ako, G. M. Hass, D. G. Grahn, and H. Neurath, Bioche,tiL~try 15, 2573 11976). 2~ H. Ako, R. J. Foster, and C. A. Ryan, Biochemisto' 13, 132 (1974). 2:, D. S. Ryan and R. E. Feeney, J. Biol. Che,t. 250, 843 (1975).
790
INHIBITORS OF VARIOUS SPECIFICITIES
[58]
Amino Acid Sequence Comparisons A comparison of the amino acid sequences of the carboxypeptidase isoinhibitors provides little insight into enzyme-inhibitor interactions. However, the carboxypeptidase inhibitors from potatoes and from tomatoes are clearly homologous and yet exhibit differences in 11 of 37 positions (Fig. 3). 4 The virtually indistinguishable Ki values of the two inhibitors for their target enzymes (carboxypeptidase A, carboxypeptidase B, S. griseus carboxypeptidase) ~ suggest that they bind through the same interactions. If this is so, nonconservative amino acid replacements probably do not occur at sites on the inhibitor that are in contact with enzyme. Figure 5 presents the amino acid sequence of the carboxypeptidase inhibitor from potatoes, with nonconservative replacements (Fig. 3) enclosed in broken lines. Residues of the inhibitor that may be chemically modified or removed without appreciable effect on inhibitory activity are indicated in Fig. 5 by solid lines. 7 Based on these data the region from Cys18 to Cys27 and the carboxyl-terminal section of the inhibitor should be of particular importance in dictating conformation or in direct interaction with enzymes.
Affinity Chromatography The affinity chromatography of pancreatic carboxypeptidases has been the object of several previous investigations. Although these procedures are effective in retaining carboxypeptidases from solution, the relatively high Ki values of the inhibitors employed (typically 0.1-10 mM) required high concentrations of ligand coupled to the matrix. At these concentrations of ligand, significant hydrophobic or ionic characteristics are imparted to the matrix, resulting in substantial nonspecific binding of protein during chromatography of crude enzyme preparations. In contrast to these substrate analogs, CPI binds tightly to target enzymes (Ki - 5 - 5 0 nM), a feature apparently retained after immobilization on Sepharose. Additional properties that render this inhibitor particularly suitable as a ligand in affinity chromatography include the following: (1) The inhibitor is stable to extremes of pH; (2) guanidine hydrochloride (6 M) does not irreversibly denature the inhibitor and thus may be used to wash the resin; (3) the amino groups of the inhibitor may be modified without reducing its activityT; thus direct coupling of CPI to CNBr-activated Sepharose is possible. This combination of tight binding and desirable characteristics has produced an affinity medium that is both effective and stable over a period of several months.
[58]
791
POTATO CARBOXYPEPTIDASE |NHIBITOR
2.0-
O--O--O--O--I
-4.0
£ E c 1.50 (3o od
v
-5.0 03 p-2.0
I.OC) Z < m n~ 0 0.5m <
I.O
t
__..__:
...... I0
Z) >_"
LO\I___ \ _r__,__-t.,_.,_,~_,_.._~./==-~.-._ 20
50
FRACTION
40
50
60
70
NUMBER
F~G. 6. C h r o m a t o g r a p h y of an extract from the visceral hump of the limpet on immobilized carboxypeptidase inhibitor. The extract was applied at p H 6.0 and the e n z y m e was eluted (,L) at pH 10. Q - - O , Absorbance at 280 nm; A - - - A , e n z y m e activity. (Details are provided in H a s s ? °)
Immobilized CPI has been used effectively in the purification of bovine and porcine carboxypeptidases A and B, 2r mast cell carboxypeptidase, 28 crayfish carboxypeptidase, '9 and carboxypeptidase A-like enzymes from the limpet 3° and from bovine placenta. 3~ An example of the use of immobilized CPI is the partial purification of limpet carboxypeptidase shown in Fig. 6. 3o After application of the crude extract and extensive elution with 0.5M NaC1-0.01 M MES (pH 6.0), the enzyme was eluted at pH 10.0 in buffer containing L-phenylalanine. This step produced a purification of over 600-fold with an 89% yield of enzymatic activity.
26 G. M. Hass and C. A. Ryan, Biochem. Biophys. Res. Commtm. 97, 1481 (1980). 27 S. P. Ager and G. M. H a s s , Anal. Biochem. 83, 285 (1977). 2~ M. T. Everitt and H. N e u r a t h , FEBS Lett. 110, 292 (1980). ._,9R. Zwilling, F. Jakob, H. Bauer, H. N e u r a t h , and D. L. Enfield, Eur. J. Biochem. 94, 223 (1979). 3o G. M. Hass, Arch. Biochem. Biophys. 198, 247 (1979). :~ J. E. D e r r - M a k u s and G. M. Hass, in preparation.
INHIBITORS OF VARIOUS SPECIFICITIES
[58]
both male and female chickens, so the synthesis of the protein is not confined to the female reproductive tract. Skin and other tissues of all the mammals examined so far contain inhibitors with properties similar to egg white cystatin, and it seems possible that these are members of a single homologous family of cysteineproteinase inhibitors. '~-'9 Common characteristics of the cystatin-type inhibitors are Mr close to 13,000, inhibition of papain and cathepsin B (with interaction even when the catalytic site is blocked), inhibition of dipeptidyl peptidase I but scarcely of bromelain, pI 4.5-6.5, and stability to heat and alkali. Amino acid sequence data will be required to confirm or deny the possibility of homology, however. 1~ K. Udaka and H. Hayashi, Biochim. Biophys. Acta 104, 600 (1965). ~7 M. J~.rvinen, Acta Chem. Stand.. Set'. B 30, 933 (1976). '~ M. Jhrvinen, J. Invest. Dermatol. 72, 114 (1978). ,9 T. Hibino, K. Fukuyama, and W. L. Epstein, Biochim. Biophys. Acta 632, 214 (1980).
[58]
Carboxypeptidase Inhibitor from Potatoes By G. MICHAEL HASS and C. A. RYAN
Although proteinaceous inhibitors of the serine proteases are widely distributed in nature, polypeptides that specifically inhibit the pancreatic carboxypeptidases have only been found in potatoes,' tomatoes," and roundworms. 3 The inhibitors from potatoes and tomatoes are highly homologous/and are believed to function both as storage proteins and as a defense mechanism by arresting digestive proteolysis of invading pests. The availability of relatively large amounts of the carboxypeptidase inhibitor from potatoes has facilitated extensive investigations of its structure and mechanism of action. In addition, affinity chromatography utilizing immobilized inhibitor has proved to be an effective and economical means of purifying a variety of metallocarboxypeptidases that are target enzymes for the inhibitor. Assay Procedure The carboxypeptidase inhibitor (CPI) is usually assayed by measuring the inhibition of carboxypeptidase A activity. The substrate most corn'
J. M. Rancour and C. A. Ryan, Arch. Biochem. Biophys. 125, 380 (1968). G. M. Hass and C. A. Ryan, Phytochemistry 19, 1329 (1980). 3 G. A. Homandberg and R. J. Peanasky, J. Biol. Chem. 251, 2226 (1976). 4 G. M. Hass and M. A. Hermodson, Plant Physiol. 65, 33 (1980).
METHODS IN ENZYMOLOGY,VOL. 80
Copyright © 1981 by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181980-9
[58]
POTATO CARBOXYPEPTIDASE INHIBITOR
779
monly employed is hippuryl-L-phenylalanine,5"~ although chloroacetyl-Ltyrosine 7 and hippuryl-DL-phenyllactate are also commonly used. A radial diffusion immunoassay for screening CPI levels in large numbers of samples has also been employed~; however, a source of anti-carboxypeptidase inhibitor antibodies is required for this procedure. Reagents
Substrate: Hippuryl-L-phenylalanine (1 mM) in 0.5 M NaCI-20 mM Tris-HC1 (pH 7.5) Enzyme: Carboxypeptidase A (Worthington or Sigma) is dissolved in 5 M NaCI-0.05 M Tris-HC1 (pH 7.5) at 4°C, and diluted with assay buffer in a concentration of approximately 0.1 mg/ml. The protein concentration can be estimated by absorbance using • E2~0nm 0 l°f¢~ = 1.88. Inhibitor: The stock inhibitor solution should be diluted with buffer such that an aliquot of 20-50 ~1 produces 25-70% inhibition. Procedure Substrate (3ml) is equilibrated at 25°C in a recording spectrophotometer. Controls are performed by adding enzyme alone (20 ~1) and monitoring the increase in absorbance at 254 nm. The inhibitor is detected as a decrease in the rate of change in absorbance. Since equilibration between enzyme, inhibitor, and substrate is rapid, there is no need for preincubation.
Data Analysis The amount of inhibitor present in the aliquot taken for assay is given by the following: CPI (tzmol) - % Inhibition CPA (/zg) 100 × 34,500/zg/tzmol where CPA is the amount of carboxypeptidase A present in the assay solution. This expression is valid for approximately 0-70% inhibition using hippuryl-L-phenylalanine as substrate and for 0-95% inhibition with chloroacetyl-L--tyrosine. The low Km for hippuryl-oL-phenyllactate and low enzyme concentration used in the assays render impractical the titration of enzymatic activity with this substrate. J. E. Folk and E. W. Schirmer, J. Biol. Chem. 238, 3884 (1963). ~;C. A. Ryan, G. M. Hass, and R. W. Kuhn, J. Biol. Chem. 249, 5495 (1974). G. M. Hass, H. Ako, D. G. Grahn, and H. Neurath, Biochemistry 15, 93 (1976). C. A. Ryan, T. Kuo, G. Pearce, and R. Kunkel, Am. Potato J. 53, 443 (1976).
780
INHIBITORSOF VARIOUSSPEC1F1CITIES I
[58]
I
12 Vo IO
• 0 ~ z
0.8 INHIBITOR I
0.6
A
m
m 04
02
i
0
2O
3 TUBE NUMBER (40 ml/tube)
FIG. 1. Chromatography of crude inhibitor I on Sephadex G-75. (See Ryan eta/. ~ for details.)
Purification Method
The purification of the principal carboxypeptidase isoinhibitor has been previously described in this series2 Briefly, this procedure involves homogenization of Russet Burbank potato tubers in H20 containing sodium dithionite, acidification to pH 3.0, ammonium sulfate fractionation (0-70% saturation), heat treatment (80c~ for 5 rain), exhaustive dialysis against H20, and lyophilization. This partially purified inhibitor (crude inhibitor I) is resolved into several zones by gel filtration on Sephadex G-75 (Fig. 1). The first inhibitor to elute from Sephadex G-75 is inhibitor I (MW -40,000'°), the second is inhibitor II (MW -20,00011), and in a "polypeptide p e a k " (fractions 77-104), there are several low-molecular-weight inhibitors of trypsin and c h y m o t r y p s i n and the carboxyY. Birk, this series, Vol. 46, p. 736. 11,j. C. Melville and C. A. Ryan, J. Bh)l. Chem. 249, 5495 (1972). ~ J. Bryant, T. R. Greene, T. Gurusaddaiah, and C. A. Ryan, Bh)chemistry 15, 3418 (1976).
[58]
POTATO CARBOXYPEPTIDASE INHIBITOR
781
peptidase isoinhibitors. Ion exchange is utilized to purify the inhibitor further. In earlier reports phosphocellulose was employed~'9; however, carboxymethyl cellulose appears to give better resolution of the many proteinase inhibitors present in the polypeptide fraction and is the preferred resin. After lyophilization, polypeptide material derived from approximately 14 kg of potatoes is dissolved in 5 mM sodium citrate (pH 4.3) and chromatographed on a column (2.5 × 40 cm) of carboxymethyl cellulose (Whatman CM-52) (Fig. 2). Following elution of the breakthrough, the column is developed with a linear gradient (2 liters total) of NaC1 (0-0.5 M) in equilibration buffer. Three zones exhibiting carboxypeptidase-inhibitory activity (Fig. 2) are observed and the three pools are lyophilized and desalted by gel filtration on Sephadex G-25. Isoinhibitor II, the predominant species, is apparently homogeneous at this stage, whereas additional chromatography steps are required to purify completely the minor isoinhibitors, CPI-I and CPI-III. v-' Yieht
The amount of carboxypeptidase inhibitor differs considerably among varieties of potatoes, ~ and it is recommended that a sampling of tubers be homogenized and assayed before engaging in a large-scale preparation. Russet Burbank potatoes grown in the states of Idaho and Washington typically yield 200 mg CPI-II, 25 mg CPI-I, and 25 mg CPI-III per 50 kg (fresh weight). Purity
The homogeneity of CPI preparations can be easily assessed by polyacrylamide-gel electrophoresis ~ or by isoelectric focusing.'2 Alternatively, the absence of leucinC in CPI is a convenient monitor that allows detection of most protein contaminants after acid hydrolysis and amino acid analysis.
Characterization Physical and Chemical Properties
CPI-I1 is an unusually stable protein easily withstanding temperatures up to 90°C and extremes of pH (pH 2-11.5) for extended time periods. Even in the tuber it is extraordinarily stable during cooking by several ~z G. M. Hass, J. E. Derr, D. J. Makus, and C. A. Ryan, Plant Phv.siol. 64, 1022 (1979).
782
INHIBITORS
OF VARIOUS SPECIFICITIES
[58]
IA[ 'IODN 0 I
0 I
"\
0 I
O CO
\ \
O tO
\ \
\ \
o
d::
-I O oa
o°
'~
E
Z
.o
.o
~
!
o w u 0~3~
io ~ o u o q J o s q v
.=-=_
[58]
783
POTATO CARBOXYPEPTIDASE INHIBITOR TABLE I CHARACTERISTICS OF CPIs FROM POTATO TUBERS Property"
CPI-I
CPI-II
CPI-III
Molecular weight Isoelectric point N-terminus K , , , , . (CPA) Ki,app. (CPB)
4089, 4219 ~' 4.6
4204, 4333 b 5.8
4075 6.5 His 1.6-3.3 n,f/ 1.3-3.3 n ~ /
" Data are taken from Hass et al. ,z b Data given for 38- and 39-residue species (see Hass et al. '~ and Fig. 2).
methods? 3 The properties of all three of the isoinhibitor species are given in Table I. Molecular weights (-3000) estimated by gel filtration are somewhat lower than the values determined by amino acid sequence analysis, presumably because the inhibitors are rather hydrophobic, and thus are retarded during chromatography. 6"12 The amino acid compositions of the carboxypeptidase isoinhibitors are given in Table II. 12 The isoinhibitors contain neither methionine nor leucine, and CPI-I lacks arginine as well. The extinction coefficient E°8}~rn = 3.0, determined by titration of stock solutions against carboxypeptidase A, is in good agreement with that predicted from the content of aromatic amino acids.
Amino Acid Sequence The principal form of the inhibitor (CPI-II) is a nearly equimolar mixture of the 38- and 39-amino acid residue peptides indicated in Fig. 3.14 This amino acid sequence, determined primarily by convential procedures, 14 has been confirmed by GC-MS analysis of suitably derivatized peptide fragments? ~ The two isoinhibitors may be separated by ionexchange chromatography, although this is not usually necessary. CPI-I differs from CPI-II at two positions (Sera0 --~ Ala and Arg32 --~ Gly). CPI-III is identical to CPI-II except that it lacks the amino-terminal pyrrolidone carboxylic acid and the adjacent glutamine residue. The six residues of half-cystine of CPI are found as 3 disulfide bonds (Cyss-Cys24, C y s 1 2 - C y s 2 7 , and C y s l s - C y s 3 4 ) . 16 ,:5 D. Y. H u a n g , B. G. Swanson, and C. A. Ryan, J. F o o d Sci. 46, 287 (1981). ,4 G. M. Hass, H. Nau, K. B i e m a n n , D. T. Grahn, L. H. Ericsson, and H. Neurath, B i o c h e m i s t r y 14, 1334 (1975). ~:' H. N a u and K. Biemann, B i o c h e m . B i o p h y s . R e s . C o m m u n . 73, 175 (1976). "~ T. R. Leary, D. T. Grahn, H. Neurath, and G. M. Hass, B i o c h e m i s t o , 18, 2252 (1979).
784
INHIBITORS OF VARIOUS SPECIFICITIES
[58]
TABLE II AMINO ACID COMPOSIrlON OF CPls Calculated no. of amino acid residues per molecule CPI-I
CPI-II
CPI-III
Amino acid
Average"
Integral
Average"
Integral
Average"
Integral
Asp Thr Ser Glu Pro Gly Ala Val Ile Tyr Phe His Lys Arg Trp ½Cys
5.00 1.91 1.04 2.66 3.25 3.74 4.83 1.27 0.85 1.08 1.13 2.01 1.89 0.01 1.69 5.75
5 2 1 2-3 3 4 5 1 1 1 1 2 2 0 1-2 6
4.96 1.95 1.98 2.42 2.94 2.96 4.00 0.98 0.90 0.99 1.03 2.15 2.13 1.05 1.3 5.8
5 2 2 2-3 3 3 4 1 1 1 l 2 2 1 1-2 6
5.00 1.92 1.75 1.15 3.28 3.02 4.18 0.95 0.95 1.1! 1.24 2.14 2.20 1.09 1.23 5.85
5 2 2 1 3 3 4 1 1 1 1 2 2 1 1-2 6
" Taken from Hass
Potato 1
Tomato 1
e t a l . v-,
His Ala His Ala
Ile ....
Asn
Lys Pro Cys
Lys
His
Gln Tyr
Val
His |Lys Pro Cys
Ser
Gln
[
!
Potato 16 ~Asp Asp Cys Ser Gly
Ala Trp IPhe Cys Gln Ala Cys Trpl Asn Ser
! Tomato 16 ~Asp Asp Cys Ser Gly
V-q
Gly Thr
IPhe
I
Cys Gln Ala Cys Trpl Arg Phe
Potato 31 IAlal Arg
Thr Cys Gly Pro Tyr Val
Tomato 31 ~
Thr Cys Gly Pro Tyr Val oH I
Gly
Gly OH
FIG. 3. A comparison of the amino acid sequences of the carboxypeptidase inhibitors from potato tubers and tomato fruit. Both 38- and 39-residue forms of CPI-II from potatoes ~2 are shown. The residues enclosed in boxes are identical in both sequences. Sequence data are taken from Hass e t a l . 4.,~
[58]
POTATO CARBOXYPEPTIDASE INHIBITOR
30
785
I0
2O
J 0 Zn
279 ~ °I
127 71
0 En
2790,.
71
FKi. 4. Stereoview of the carboxypeptidase inhibitor in association with carboxypeptidase A as determined by X-ray diffraction at 2.5 A, resolution. ~
The amino acid sequence of CPI demonstrates that it is homologous to the carboxypeptidase inhibitor from tomato fruit (Fig. 3), and suggests that it may be homologous to a polypeptide chymotrypsin inhibitor from potatoesJ 7 as well as to several toxic peptides including viscotoxin A2 from the European mistletoe and/J-purothinine from wheatJ 8 although these latter comparisons are highly speculative and are based primarily on the half-cystine residues. Three-Dimensional Structure
The structure of CPI-II has been deduced by X-ray diffraction analysis at 2.5 A resolution of the inhibitor-carboxypeptidase A complex? 9 CPI-II has neither helical structures nor/J-pleated sheets, as is seen in Fig. 4. X - R a y Diffraction Analysis
The interaction of the carboxyl-terminal region of the inhibitor from potatoes with carboxypeptidases has been clearly indicated by X-ray crystallographic analysis of the enzyme-inhibitor complex at 2.5 A resolution TM (Fig. 4). That report demonstrates that the inhibitor binds like an extended substrate. The most unusual features of the inhibitor-enzyme ~7 G. M. Hass, R. Venkatakrishnan, and C. A. Ryan, Proc. Natl. Acad. Sci. U.S.A. 73, 1941
(1976). ~ D. J. Strydom,J. Mol. Evol. 9, 349 (1977). ~ D. C. Rees and W. N. Lipscomb,Pro('. Natl. Acad. Sci. U.S.A. 77, 4633 (1980).
786
INHtBITORS OF VARIOUS SPECIFICITIES
[58]
interaction are that the Val38-Gly39 peptide bond is cleaved in the complex, and that GIy39 appears to be trapped in the binding pocket of the enzyme. 19 The comparison of the amino acid sequences of the inhibitors from tomatoes and potatoes (Fig. 3) is of particular interest in this respect. The amino acid residue that would correspond to Gly39 of the inhibitor from potatoes is not found in the inhibitor from tomatoes. This observation has led to recent studies demonstrating that carboxypeptidase A rapidly removes Gly39 from the potato inhibitor and that this residue is not required for enzyme-inhibitor interaction. 26 The X-ray diffraction analysis of the enzyme-inhibitor complex 19 indicates that residues 37-39 of the inhibitor interact extensively with the enzyme active site, an observation consistent with the complete homology in this region between the inhibitor from potatoes and from tomatoes (Fig. 3). Additional enzyme-inhibitor contacts involve the rings of Trp28, Phe22, and, to a lesser extent, HiSl~. Of these residues the only replacement observed in His15 ~ Gln (Fig. 3). The final enzyme-inhibitor contact involves the backbone of Trp~s-Ser30 of the inhibitor and Ile24T, Tyr24s, and Lys~9 of the enzyme. Although Trp28 is conserved, nonconservative replacements are found at residues 29 and 30. Apparently, these replacements do not significantly affect the conformation of the polypeptide backbone in this region. 1~ The structure is extremely compact with the disulfide bond joining Cys,s and C y s 3 4 passing through a closed loop that involves the other disulfide bridges. The structure indicated that the Val~8 and Gly39 bond was broken but that the GIy39was trapped in the specificity pocket of the enzyme (see later). Unfortunately, a structure based on X-ray diffraction of crystals of free inhibitor is not yet available. Thus the possibility that conformation changes occur in the inhibitor other than the breaking of the Va138-Gly39bond on binding enzyme has not been investigated.
Inhibition Spectrum CPI was first detected as an impurity in crystals of inhibitor I by its ability to inhibit porcine carboxypeptidase B. ''2° The inhibitor was purified and demonstrated to affect bovine carboxypeptidase A. 6 It has now been tested against a variety of carboxypeptidases from animal, plant, fungal, and bacterial sources. 21 The association between each enzyme and CPI was monitored either by inhibition of enzymatic activity ([CPI] approx. 1-10 nM) or by the binding of enzyme to CPI-Sepharose. Enzymes that participate in the degradation of dietary protein were par~ C. A. Ryan, Biochemist13' 5, 1592 (1966). .2, G. M. Hass, S. P. Ager, and D. J. Makus, Fed. Proc., Fed. Am. Soc. Exp. Biol. 39, 1689 (1980).
[58]
787
POTATO CARBOXYPEPTIDASE INHIBITOR T A B L E III BINDING OF CP1 TO DIGESTIVE TRACT CARBOXYPEPTIDASES AND TO THEIR ZYMOGENS a Procedure
Enzyme
Inhibition of e n z y m a t i c activi t y
Bovine CPDase A Bovine CPDase B Porcine CPDase A Porcine CPDase B Shrimp CPDase A Shrimp CPDase B Lungfish CPDase B Crayfish CPDase Cab bage looper C P D a s e L i m p e t CPDase Bovine Pro-CPDase A Bovine Pro-CPDase B Porcine Pro-CPDase A Porcine Pro-CPDase B Lungfish Pro-CPDase B
+ + + + + + + + + + NA ~ NA NA NA
Binding to CPI-Sepharose + + + + ND b ND ND + ND +
-
" Data are taken from Hass et al. ~ ~' ND, not determined. " NA, not applicable.
tially purified from animal species as diverse as the cow and the limpet, and all were potently affected by the inhibitor (Table III). H o w e v e r , several zymogens of the enzymes in this group were tested and shown not to bind immobilized inhibitor (Table III). With the exception of an enzyme from mast cells and a novel carboxypeptidase A-like enzyme from bovine placenta, all animal carboxypeptidases that were not of digestive tract origin were not affected by the inhibitor (Table IV). The inhibitor had no effect on the enzymatic activities of all plant and most microbial carboxypeptidases (Table V) tested. H o w e v e r , a strong association between the inhibitor and Streptomyces griseus carboxypeptidase has been noted, 22 and a low affinity (K~ - 1 0 -5 M) for a carboxypeptidase G~ from an Achtetobacter was found as well. This inhibition spectrum not only elucidates possible evolutionary relationships among the carboxypeptidases, but also identifies enzymes whose purification might be facilitated by using immobilized inhibitor as described next. ~ L. Gage-White, B. E. Hunt, G. M. Hass, and W. M. Awad, Jr., Fed. Proc.. Fed. Am. Soc. Exp. Biol. 36, 892 (1977).
788
INHIBITORS OF VARIOUS SPECIFICITIES
[58]
T A B L E IV BINDING OF CPl ~o INTRACELLULAR AND REGULATORY CARBOXYPEPTIDASES"
Procedure
Enzyme
Inhibition of
Binding of
enzymatic activity
CPI-Sepharose
Mast cell CPDase (rat) Placenta CPDase (bovine) Cathepsin A (bovine spleen) Catheptic CPDase B (bovine spleen) CPDase P (bovine kidney) CPDase N (human, bovine, porcine serum) CPDase (chicken muscle) Kininase II
+
N D t, ND ND ND ND
" Data are taken from Hass eta/.'-" ~' ND, not determined.
Mechanism of Action The inhibition of bovine carboxypeptidase A and porcine carboxypeptidase B is competitive with K~ values of approximately 5 and 50 riM, respectively. 6 Because CPI binds much tighter than model substrates to the carboxypeptidases and because CPI represents a new class of inhibitors, much effort has been directed at probing the mode of binding of this inhibitor. TABLE V BINDING OF CPI l o MICROBIAL CARBOXYPEPTIDASES 't
Procedure
Inhibition of
Binding to
Enzyme
e n z y m a t i c activity
CPI-Sepharose
CPDase GI (P. ~t,tzeri) CPDase G1 (Acinetobacter sp.) CPDase G1 (Flal'oba('terium sp.) S. g,'ise,s CPDase Yeast protease ~ Yeast protease Y P. omnivorum CPDase S. ,~clerotiorllm CPDase
+ + +
" Data are taken from Hass et al.'-" t, ND, not determined.
-
ND ~ + ND ND ND ND
[58]
1
Pro Ile Cys I Asn l
......
16
31
789
POTATO CARBOXYPEPTIDASE INHIBITOR
Cys Ser Gly Ala
Ala
Pro Cys
Thr
%__,
Phe Cys Gln Ala Cys
I Asn Ser I f L .... .J
C" . . . . I I Thr Cys Gly Pro Tyr Val 11Gly OH I I. . . . .
I
FIG. 5. A m i n o acid residues of the carboxypeptidase inhibitor from potatoes lhat do not contribute significantly to the free energy of association of the e n z y m e - i n h i b i t o r complex are enclosed in boxes. Evidence is based on a comparison with the sequence o f the inhibitor from tomatoes (___)4 and chemical modification studies ( ).7
Chemical Modification Studies The interaction in solution of CPI with target enzymes has been investigated by modifying either the inhibitor or the enzyme. First, several chemical derivatives of the inhibitor were prepared and their Ki values with respect to carboxypeptidases A and B determined to evaluate the effect of each modification on enzyme-inhibitor interaction£ Residues I-5 could be removed, the side-chain carboxylate groups modified, lysines at positions 10 and 13 acetylated, or the arginine at position 31 reacted with phenylglyoxal without affecting the strength of binding to either carboxypeptidase. Tyrosine-37 and the alpha-carboxylate of glycine-39, however, appear to be involved in contact with target enzymes (Fig. 5). In all cases, parallel effects of a given modification on the binding to both carboxypeptidases were observed, indicating that similar or identical contacts between enzyme and inhibitor were utilized in both systems.7 A second study of CPI binding to derivatives of carboxypeptidase A revealed that the binding of the inhibitor to arsanilazocarboxypeptidase A produced spectral changes similar to those observed when typical competitive inhibitors bind to this enzyme derivative. 23 Enzymatic activity was not required for association with inhibitor, since apocarboxypeptidase bound inhibitor. This situation was analogous to the tight association between anhydrotrypsin and anhydrochymotrypsin and their respective proteinase inhibitors.24 On the other hand, blockage of the binding pocket of carboxypeptidase A by reaction with N-bromoacetyl-N-methyl-L-phenylalanine had little effect on the free energy of association with inhibitor, whereas a similar modification of trypsin or chymotrypsin greatly weakens their interactions with proteinase inhibitors. ~ 2:~ H. Ako, G. M. Hass, D. G. Grahn, and H. Neurath, Bioche,tiL~try 15, 2573 11976). 2~ H. Ako, R. J. Foster, and C. A. Ryan, Biochemisto' 13, 132 (1974). 2:, D. S. Ryan and R. E. Feeney, J. Biol. Che,t. 250, 843 (1975).
790
INHIBITORS OF VARIOUS SPECIFICITIES
[58]
Amino Acid Sequence Comparisons A comparison of the amino acid sequences of the carboxypeptidase isoinhibitors provides little insight into enzyme-inhibitor interactions. However, the carboxypeptidase inhibitors from potatoes and from tomatoes are clearly homologous and yet exhibit differences in 11 of 37 positions (Fig. 3). 4 The virtually indistinguishable Ki values of the two inhibitors for their target enzymes (carboxypeptidase A, carboxypeptidase B, S. griseus carboxypeptidase) ~ suggest that they bind through the same interactions. If this is so, nonconservative amino acid replacements probably do not occur at sites on the inhibitor that are in contact with enzyme. Figure 5 presents the amino acid sequence of the carboxypeptidase inhibitor from potatoes, with nonconservative replacements (Fig. 3) enclosed in broken lines. Residues of the inhibitor that may be chemically modified or removed without appreciable effect on inhibitory activity are indicated in Fig. 5 by solid lines. 7 Based on these data the region from Cys18 to Cys27 and the carboxyl-terminal section of the inhibitor should be of particular importance in dictating conformation or in direct interaction with enzymes.
Affinity Chromatography The affinity chromatography of pancreatic carboxypeptidases has been the object of several previous investigations. Although these procedures are effective in retaining carboxypeptidases from solution, the relatively high Ki values of the inhibitors employed (typically 0.1-10 mM) required high concentrations of ligand coupled to the matrix. At these concentrations of ligand, significant hydrophobic or ionic characteristics are imparted to the matrix, resulting in substantial nonspecific binding of protein during chromatography of crude enzyme preparations. In contrast to these substrate analogs, CPI binds tightly to target enzymes (Ki - 5 - 5 0 nM), a feature apparently retained after immobilization on Sepharose. Additional properties that render this inhibitor particularly suitable as a ligand in affinity chromatography include the following: (1) The inhibitor is stable to extremes of pH; (2) guanidine hydrochloride (6 M) does not irreversibly denature the inhibitor and thus may be used to wash the resin; (3) the amino groups of the inhibitor may be modified without reducing its activityT; thus direct coupling of CPI to CNBr-activated Sepharose is possible. This combination of tight binding and desirable characteristics has produced an affinity medium that is both effective and stable over a period of several months.
[58]
791
POTATO CARBOXYPEPTIDASE |NHIBITOR
2.0-
O--O--O--O--I
-4.0
£ E c 1.50 (3o od
v
-5.0 03 p-2.0
I.OC) Z < m n~ 0 0.5m <
I.O
t
__..__:
...... I0
Z) >_"
LO\I___ \ _r__,__-t.,_.,_,~_,_.._~./==-~.-._ 20
50
FRACTION
40
50
60
70
NUMBER
F~G. 6. C h r o m a t o g r a p h y of an extract from the visceral hump of the limpet on immobilized carboxypeptidase inhibitor. The extract was applied at p H 6.0 and the e n z y m e was eluted (,L) at pH 10. Q - - O , Absorbance at 280 nm; A - - - A , e n z y m e activity. (Details are provided in H a s s ? °)
Immobilized CPI has been used effectively in the purification of bovine and porcine carboxypeptidases A and B, 2r mast cell carboxypeptidase, 28 crayfish carboxypeptidase, '9 and carboxypeptidase A-like enzymes from the limpet 3° and from bovine placenta. 3~ An example of the use of immobilized CPI is the partial purification of limpet carboxypeptidase shown in Fig. 6. 3o After application of the crude extract and extensive elution with 0.5M NaC1-0.01 M MES (pH 6.0), the enzyme was eluted at pH 10.0 in buffer containing L-phenylalanine. This step produced a purification of over 600-fold with an 89% yield of enzymatic activity.
26 G. M. Hass and C. A. Ryan, Biochem. Biophys. Res. Commtm. 97, 1481 (1980). 27 S. P. Ager and G. M. H a s s , Anal. Biochem. 83, 285 (1977). 2~ M. T. Everitt and H. N e u r a t h , FEBS Lett. 110, 292 (1980). ._,9R. Zwilling, F. Jakob, H. Bauer, H. N e u r a t h , and D. L. Enfield, Eur. J. Biochem. 94, 223 (1979). 3o G. M. Hass, Arch. Biochem. Biophys. 198, 247 (1979). :~ J. E. D e r r - M a k u s and G. M. Hass, in preparation.