VOL.
8,
NO.
12,
DECEMBEK
1969
bacher, L. J., Feder, J., Gunter, C. R., KCzdy, F. J., Killheffer, J. V., Jr., Marshall, T. H., Miller, C. G., Roeske, R. W., and Stoops, J. K. (1966), J. Am. Chem. Soc. 88,5890. Bender, M. L., and Marshall, T. H. (1968), J. Am. Chem. SOC. 90,201. Bingle, J. P., and Czerkawski, J. W. (1963), Biochem. J. 87, 34P. Czerkawski, J. W., and Bingle, J. P. (1963), Biacliem. J. 87, 33. Dvonch, W., and Alburn, H. E. (1959), Arch. Biochem. Biophys. 79,146. Hall, D. A. (1953), Biochem. J . 55, XXXV. Hall, D. A. (1957), Arch. Biochern. Biopliys. 67,366. Hall, D. A. (1964), Elastolysis and Aging, Springfield, Ill., C. C. Thomas. Hormann, H., and Fujii, T. (1962), 2.Physiol. Chem. 328, 65. Kunitz, M. (1947),J. Gen. Physiol. 30,291. Lamy, F., Craig, C. P., and Tauber, S. (1961), J . Biol. Chem. 236, 86. Layne, E. (1957). Methods Enzjxiol. 3,447.
Lewis, U. J., and Thiele, E. H. (1957), J. Am. Clienz. Soc. 79, 755. Lewis, U. J., Williams, D. E., and Brink, N. G. (1956), J . Biol. Chem. 222,705. Ling, V., and Anwar, R. A. (1966), Biochem. Biopliys. Res. Commun. 24,593. Loeven, W. A. (1963), Acta Physiol. Pharmacol. Need. 12, 57. Long, C. (1961), Biochemist’s Handbook, Princeton, N. J., D. van Nostrand, p 30. Mandl, I. (1962), Methods Enzymol. 5,665. Naughton, M. A.: and Sanger, F. (1961), Biochern. J. 78, 156. Report of the Commission on Enzymes of the International Union of Biochemistry (1961), London, Pergamon, p 7. Smillie, L. B., and Hartley, B. S . (1964), J. Mol. Biol. 10, 183. Solyom, A., Borsy, J., and Tolnay, P. (1964), Biochem. Phurinacol. 13, 391. Sblyom, A., and Tolnay, P. (1965), Enzymologiu 28,52. Uriel, J., and Avrameas, S. (1965), Biochemistr)~4 , 1740. Walford, R. L., and Kickhofen, B. (1962), Arch. Biochem. Biopliys. 98, 191.
Porcine Elastase. 11. Properties of the TyrosinateSplitting Enzymes and the Specificity of Elastase* Thomas H. Marshal1,t John R. Whitaker,$. and Myron L. Benders
Three enzymes catalyzing the hydrolysis of p-nitrophenyl N-benzyloxycarbonyl-L-tyrosinate were partially purified from elastoproteinase by chromatography on DEAESephadex A-50 at pH 8.8. The chromatographed enzymes showed complex kinetics including activation by acetate ion, acetonitrile, and reaction products. When the tyrosinatesplitting enzymes were assayed without having been separated from elastase, they exhibited normal kinetics. The tyrosinate enzymes were distinguished from elastase by (1) high activity against p-nitrophenyl N-benzyloxycarbonyl-L-tyrosinate,(2) ABSTRACT:
P
orcine pancreas has been shown to contain three enzymes with high specific activity against p-nitrophenyl N-benzyloxycarbonyl-L-tyrosinate (Marshall et ul., 1969). Preparations of crystalline elastase, including those prepared in these laboratories and those obtained commercially, contained
* From the Department of Chemistry, Northwestern University, Evanston, Illinois 60201, and the Department of Food Science and Technology, University of California, Davis, California 95616. Receiced Airgust 25, 1969. Abstracted in part from the Ph.D. thesis of Thomas H. Marshall, Northwestern University, 1967. 1’ Present address: Department of Chemistry, Northern Illinois University, DeKalb, Ill. 601 15. $ Department of Food Science and Technology, University of California, Davis, Calif. 95616. $ Department of Chemistry, Northwestern University, Evanston, Ill. 60201.
greater sensitivity to inhibition by soybean trypsin inhibitor, and (3) slower rates of reaction with diethyl p-nitrophenyl phosphate. The tyrosinate enzymes appear to be different from all the other known esterases of pancreas. The specificity of the tyrosinate enzymes msy have possible significance in relation to the transitory nature of the dityrosine crosslinkage in fetal elastin. The specificity of elastase was measured against eight substrates and compared with that of a-chymotrypsin and hydroxide ion.
variable amounts of these enzymes. These tyrosinate enzymes are distinct from elastase which has very little if any activity against this substrate. The tyrosinate-splitting enzymes can be partially removed from elastase by electrophoretic purification of crystalline elastase or by repeated chromatography on CM-cellulose or on DEAE-Sephadex A-50, particularly at alkaline pH. In this paper we report some properties of the tyrosinate enzymes which may be used to distinguish them from elastase. We also report on the specificity of elastase as measured against eight substrates and as compared with the specificity of a-chymotrypsin and hydroxide ion. The comparative specificities of elastase and a-chymotrypsin are of interest since the X-ray studies now in progress (Shotton et ai., 1968) should reveal in detail the differences in their three dimensional structures.
PO KC IN^ E L A S T A S E .
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4671
BIOCHEMISTRY
I
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1
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Medium Effects upon the Enzyme-Catalyzed Hydrolysis of p-Nitrophenyl N-Benzyloxycarbonyl-L-tyrosinate..
TABLE I:
2.0
Enzyme Concn (mg of Protein/ml) X IO2 pH
1.8
?:N 5
1.6
W W
e
1.4
(3
9
i 2
4
6
8
1
0
TIME (SEC)
FIGURE1: Reaction of elastase with a large excess of diethyl p-nitrophenyl phosphate. (A) Release of p-nitrophenol and (B) loss of activity against p-nitrophenyl N-benzyloxycarbonyl-t-tyrosinate. Worthington electrophoretically purified elastase lots 6507B and 5691/923 (pH 7.7), 7 % (v/v) acetonitrile-water, 25", SO= 7.5 X 10-3 M.
Materials and Methods The materials and methods used in this investigation were identical with those reported in the accompanying publication (Marshall et al., 1969). The concentration of a-chymotrypsin was determined by titration with N-trans-cinnamoylimidazole(Schonbaum et al., 1961). The saponification rate constants were measured under pseudo-first-order conditions using a large excess of NaOH. The second-order rate constant was derived from the slope of a plot of the observed first-order constant us. hydroxide ion concentration.
105 M
x
k
102
sec-'
1.03
7.74
Phosphate,
1.5
0.609
2.14 5.31 10.5 4.25
7.73 7.67 7.58 6.42
I I I I I
1.5 1.5 1.5 1.5
1.17 3.10 6.00 1.02 (0.46)b
4.25
5.97
Acetate,
1.5
1.35
1.5
0.43 (0.35)b
1.2
I .o
so x Buffer
I 4.25
5.97
= = = = =
=
0.05 0.05 0.05 0.05 0.05
0.05
Citrate,
I
=
0.05
4.25
5.86
Acetate,.
0.75
0.96 (1.28)b
4.25
5.88
I I
1.5
0.88 (1.17)b
4.25
5.78
Acetate,c
0.75
0.615 (l.Ol)*
0.75
0.445 (0.71)b
I 4.25
5.80
= =
=
0.10 0.10
0.60
Phosphatep
I
= 0.60
(v/v), 25.0 '. Worthington elasa Acetonitrile-water 0.7 tase 5691/923 was made up as a stock solution in 0.1 M acetate buffer (pH 4.6). The experimental rate constant is corrected to pH 6.0 according to the pH dependence for this activity shown in Figure 2 to facilitate comparison. cKCI added to increase ionic strength.
Results Reaction of the Tyrosinate Enzymes with Diethyl p-Nitrophenyl Phosphate. The tyrosinate enzymes can be distinguished from both elastase and a-chymotrypsin by their different rates of inhibition with diethyl p-nitrophenyl phosphate. When electrophoretically purified elastase (Worthington 6507B and 5691/923) was reacted with diethyl p-nitrophenyl phosphate, it lost its activity against p-nitrophenyl N-benzyloxycarbonylglycinate and p-nitrophenyl trimethylacetate at the same rate at which nitrophenol was liberated from diethyl p-nitrophenyl phosphate. However, the rate of loss of activity against p-nitrophenyl N-benzyloxycarbony1-Ltyrosinate was some 20 times slower than the rate of liberation of p-nitrophenol from diethyl p-nitrophenyl phosphate (Figure 1). a-Chymotrypsin lost activity against p-nitrophenyl N-benzyloxycarbonyl-L-tyrosinateat the same rate as it reacted with diethyl p-nitrophenyl phosphate. p-Nitrophenyl N-Benzyloxycarbonyl-L-tyrosinate Hydrolysis Catalyzed by Electrophoretically Purified Elastase. We have demonstrated above that the tyrosinate enzymes in elastase preparations can be distinguished from elastase. The effect of several parameters on the rate of hydrolysis of p-
4672
MARSHALL,
WHITAKER,
AND BENDER
nitrophenyl N-benzyloxycarbonyl-L-tyrosinate was studied in some detail using a preparation of electrophoretically purified elastase in which the tyrosinate enzymes were present in trace amounts. Our data indicate that elastase makes no contribution to the hydrolysis of the substrate. The results of several experiments under various conditions are presented in Table I. The reactions are all first order in substrate concentration. This can be explained assuming that K, >> So. The first-order rate constant is independent of initial substrate concentration and is not affected by initial product equal to substrate concentration. Thus the reaction is truly first order and not apparent first order due to product inhibition (Whitaker et al., 1966). The first order rate constant is directly proportional to concentration of total protein over a tenfold range. A minor buffer effect is evident in that the rate constant is 2.5 to 3.0 times greater in the presence of acetate buffer than in the presence of phosphate, citrate, or Tris buffers. The difference is reduced at higher salt concentrations. Enzyme catalysis is abolished in 7 M urea. The pH dependence
VOI..
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12,
NO.
3
1969
DECEMBER
4
5
6
7
8
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2: The pH-rate profile for the enzyme catalyzed hydrolysis of p-nitrophenyl N-benzyloxycarbonyl-L-tyrosinate,25.0", 0.83% (v/v) acetonitrile-water, I = 0.05, phosphate buffer above pH 5.6 and citrate below pH 5.6. Worthington elastase lot 5691/923; stock solution prepared in 0.1 M acetate buffer (pH 4.6). Total protein concentration ranged from 0.22 to 0.056 mg per ml. The first-order rate constant is corrected for spontaneous hydrolysis and normalized to a total protein concentration of 0.056 mg/ml. The dashed line represents a theoretical curve based on a pK, of 6.6 and a maximal limitsec-'. ing rate constant of 3.2 X
0
1
3 TIME ( M I N I
5
FIGURE
of the reaction is presented in Figure 2. The data conform well to a theoretical curve based on a kinetic pK, of 6.6 and a maximal limiting rate constant of 3.2 X 10+ sec-I. p-Nitrophenyl N-Benzyloxycarbonyl-L-tyrosinateHydrolysis Catalyzed by Fractions from Chromatographed Elastoproteinase. Elastoproteinase was purified as described in Figures 3 and 4 of the accompanying publication (Marshall et al., 1969) in which we have shown that chromatography of elastoproteinase (prepared by the method of Loeven, 1963) on DEAE-Sephadex A-50 gives a t least five components with activity against casein. Associated with the first peak with activity against casein are two components with tyrosinate activity and one with elastase activity. These components overlap each other but are distinct chromatographically. The second component with activity against casein is chromatographically identical with a third, and major, component with tyrosinate activity. The components with tyrosinate activity will be designated as tyrosinate enzymes I (peak fraction with highest activity, no. 19; see Figure 4, Marshall et al., 1969), I1 (peak fraction with highest activity, no. 28), and I11 (peak fraction with highest activity, no. 50). The fraction with highest activity against elastin-orcein was no. 23 and was contaminated with tyrosinate enzymes I and 11. The hydrolysis of p-nitrophenyl N-benzyloxycarbony1-Ltyrosinate under various conditions by two distinct chromatographic components of elastoproteinase (fractions 23 and 50) is presented in Figure 3. The kinetics of the reaction catalyzed by the enzymes in fraction 23 were normal even though the tyrosinate activity was 1000 times too large to be due t o elastase (curve A). However, the reaction with tyrosinate enzyme I11 (fraction 50) was complex (curve B). The reaction accelerated with increasing product concentration. Initial product concentration equal t o initial substrate concen-
3 : Rate of hydrolysis of p-nitrophenyl N-benzyloxycarbonylL-tyrosinate under various conditions. The reaction was carried out at pH 8.0 in 0.01 M Tris-HC1 buffer at 25" and was started in all cases by addition of substrate (final concentration, 5.91 X M) to the reaction cuvet. Reaction volume was 3.05 ml. The enzyme fractions referred to are those of Figure 4 (Marshall et ai., 1969). The meaning of the curves are: (A) 0.02 ml of enzyme (0.0394 mg) from fraction 23 assayed in the presence of 2.4 and of 12.2% acetonitrile, (B) 0.02 ml of enzyme (0.00438 mg) from fraction 50 assayed in the presence of 2.4% acetonitrile, ( C )immediately on completion of reaction in case B new substrate added to cuvet (final concentration of acetonitrile, 4.8 %), (D) 0.02 ml of 1:10 dilution of enzyme (4.38 X mg) in fraction 50 assayed in the presence of 2.4% acetonitrile, and (E) reaction conditions as in case Dcexcept 12.2% acetonitrile used. Protein concentration based on Et& 12.1.
FIGURE
tration resulted in normal kinetics and increased initial rate (curve C ) . A strong activation by acetonitrile occurred with this fraction and the kinetics were normal at sufficiently high acetonitrile concentration (curve E). This marked activation by acetonitrile is indicated by the following data: the initial rates of hydrolysis at 2.4, 8.95, 12.2, and 15Sz acetonitrile and constant enzyme concentration (4.38 X mg/3 ml) were (curve D), 12.9 x 28.8 x loF8,and 28.2 X 2.41 x 10-8 M sec-1, respectively. Other fractions with elastase activity (fractions 20-25) showed normal kinetics and no activation by acetonitrile. Tyrosinate enzyme I (fraction 19) and tyrosinate enzymes I1 and I11 (fractions 26-64) gave complex kinetics but in the presence of 12.2% acetonitrile normal kinetics and a n enhanced rate were observed. The Reaction of p-Nitrophenyl Trimethylacetate with the Tyrosinate Enzymes. Elastase hydrolyzes p-nitrophenyl trimethylacetate (Bender and Marshall, 1968). The tyrosinate enzymes are inhibited by this substrate in a time-dependent process. Electrophoretically purified enzyme (Worthington 5691) was incubated with p-nitrophenyl trimethylacetate in a cuvet in the spectrophotometer cell compartment and then a n aliquot of p-nitrophenyl N-benzyloxycarbonyl-L-tyrosinate was added. The rate constant for the resulting first-order reaction decreased with increasing period of prior incubation with p-nitrophenyl trimethylacetate. The results are presented in Figure 4. The inhibition is probably due to a n acylation reaction similar to the inhibition of a-chymotrypsin by p-nitrophenyl trimethylacetate (McDonald and Balls, 1957). It
P O R C I N E ELASTASE.
11
4673
B 1 0 C tI E M I ST R Y
"/% a
01 0
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7
OY 0
FIGURE 4 : Inhibition of activity against p-nitrophenyl N-benzyloxycarbonyl-L-tyrosinate by p-nitrophenyl trimethylacetate. Elastase 5691 (Worthington; 3.3 X M) was incubated with 1.2 X 10-4 M inhibitor in 0.1 M phosphate buffer (pH 7.8), containing 3.1 (v/v) acetonitrile. Activity was assayed by adding 1.5 X M substrate.
appears that one or more of the three tyrosinate enzymes must be inhibited by reaction with p-nitrophenyl trimethylacetate but that the activity of one or more of these enzymes is not affected by this compound. The residual activity left after maximum inhibition is reached is too high to be due t o elastase only. The Inhibition of Tyrosinate Enzymes by Soybean Trypsin Inhibitor. The activity of elastase can be distinguished from that of the tyrosinate enzymes by the latter's greater sensitivity to inhibition by soybean trypsin inhibitor. At a n inhibitor concentration twice that sufficient to reduce its tyrosinate activity to less than 4% of the original value, an electrophoretically purified elastase (Worthington 6505) retained 80 of its original activity against p-nitrophenyl N-benzyloxycarbonylglycinate and 70 % of its original activity against p-nitrophenyl trimethylacetate. This cannot be due to inability of the inhibitor to displace these latter two substrates from the enzyme because one of them (trimethylacetate) binds strongly and the other (glycinate) is bound weakly to the
-11
I
I
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I
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I
1
loot
I I
INHIBITOR
I
IO CONC.
30 (MGIML
I
x
50 103)
FIGURE 5 : Inhibition of p-nitrophenyl N-benzyloxycarbonyl-L-tyrosinate activity by soybean trypsin inhibitor. SO= 1.5 X M (pH 7 . 7 9 , phosphate buffer, I = 0.05. Worthington electrophoretically purified elastase (lot 6505) was used at concentration of 0.0374 mg/ml in reaction mixture. Stock solutions of Mann soybean trypsin inhibitor were prepared in pH 7.8 phosphate buffer, I = 0.05. In absence of inhibitor kobsd was 1.25 X sec-l. The enzyme activity has been corrected for spontaneous hydrolysis by inhibitor.
4674
MARSHALL,
WHITAKER,
A N D
20
BENDER
I
I
0.1 INHIBITOR
0.2 CONC.
I
0.3 (MG/ML)
I
0.4
6: Inhibition of elastase activity against p-nitrophenyl Nbenzyloxycarbonylglycinate by soybean trypsin inhibitor. CondiM, SO, tions: 0.075 mg/ml of Worthington 6505 elastase,9.4 X and pH 7.7. FIGURE
enzyme under the conditions employed. p-Nitrophenyl N-benzyloxycarbonyl-L-tyrosinateis also bound weakly t o the enzymes. The relative strengths of binding referred t o above follow from the fact that So/K, (app) is five for p-nitrophenyl trimethylacetate and that p-nitrophenyl N-benzyloxycarbonyl-L-tyrosinate and p-nitrophenyl N-benzyloxycarbonylglycinate exhibit first-order kinetics, implying that So
