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Catalytic activity of chymotrypsin in crystalline state
364
SHORT COMMUNICATIONS
In summary, the 50% ammonium sulfate precipitate from a homogenate of adrenal cortical tissue showed only 2oa-hydroxysteroid dehydrogenase activity. This study was supported by Grant AM-o3269, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U.S. Public Health Service.
Departments of Endocrinology and Biochemistry, Southwest Foundation for Research and Education, San Antonio, Texas (U.S.A.)
C. MATTHIJSSEN J. E. MANDEL
P. T. SEIDEN
x C. MATTHIJSSEN AND J. E. MANDEL, Biochim. Biophys. Acta, 82 (1964) 138. 2 L. R. AX~LROD, J. Biol. Chem., 2o 5 (1953) 173. 3 A. ZAFFARONI AND B. BURTON, J. Biol. Chem., 193 (1951) 749. 4 L. R. AXELROD, Anal. Chem., 27 (1955) 13o5. s W. G. WIEST ANn R. B. WILCOX, J. Biol. Chem., 236 (1961) 2425.
Received March 3Ist, 1964
Biochim. l~iophys. Acta, 89 (1964) 363-364
sc 63048
Catalytic activity of chymotryp$in in crystalline state Enzyme molecules generally function in dilute aqueous solution. However, it would be interesting to know the relationship between a protein molecule in solution and one in the crystalline state 1. It is now possible by means of X-ray diffraction to establish the complete three-dimensional structure of protein molecule in this latter state, as illustrated in the case of myoglobin 2,s. With this in view, a comparison of the enzymic activity of protein molecules in the crystalline state with those in solution was undertaken. Chymotrypsin (EC 3.4.4.5) in the crystalline state was found to be catalytically active with enzymic properties similar to those obtained in dilute solution .The enzymic activity of the crystals was measured by using acetyl-L-tyrosine hydrazide and acetyl-L-tyrosine ethyl ester as substrates * in 8o% saturated (NH4),SO ,. a-Chy{-notrypsin (Mann, salt-free, 3XCryst) was crystallized at room temperature from 0.5 s~turated (NH4),SO 4 yielding crystals approx, o.i-o.2 mm in width and 0.5-0.8 mm in length. A typical enzyme-assay was run as follows : crystals of chymotrypsin (I mg) were suspended in a solution (4 ml) of 2. IO-3 M acetyl-L-tyrosine hydrazide substance in 80% saturated (NH4)2SO , at pH 7. The reaction mixture was incubated at 25 ° with mild shaking. After a given time interval (30 min), crystals were allowed to settle for about a minute and aliquots of the supernatant solution (o.25 ml) were withdrawn and the hydrazine liberated was determined colorimetrically by the use of Ehrlich's Reagent. Results were compared with those obtained with a standard chymotrypsin solution to which 80% saturated (NH4),SO 4 had to be added. (The presence of the 80% saturated (NH4),SO , in the solution increased the rate of enzymic hydrolysis by 25-30%. ) The weight of the solid enzyme after the assay was determined spectrophotometricaUy* by measuring tile protein concentration of the dissolved crystals. A control experiment was performed to determine the solubility of
Biochim. Biophys. Aaa, 89 (1964) 364-366
365
SHORT COMMUNICATIONS
the enzyme crystals in the assay mixture by measuring the enzymic activity and protein concentration of the 80% saturated (NH4)~SO ~ supernatant solution of the crystals. The solubility of the crystals (approx. o.0o8 mg in solution for I mg of crystalline enzyme) was found to be less than 1%. This could thus only account for a small fraction of the observed activity. All results expressing enzymic activity of chymotrypsin in crystalline state were corrected accordingly. From this assay, the rate of hydrolysis of acetyl-e-tyrosine hydrazide by I mg of the crystals of chymotrypsin was equal to the activity of o.18 mg of chymotrypsin in solution or I 8 % activity as compared with the same amount of enzyme in solution. The activity was found to vary from I5-2I % depending on the different batches of 0.7 0.6 0.5
E•0.4
U3 tO ~- 0.3
~ o.2 0
~ o.1
<
0
o
I
4.0 1.o 2'.0 3'.0 ' Crystals of chymotvypsin (rag pep sample)
5t.0
I
6.0
Fig. I. R a t e of hydrolyses of acetyl-L-tyrosine hydrazide s u b s t r a t e as a function of the a m o u n t of crystalline c h y m o t r y p s i n p r e s e n t in the solid phase in 8 o % s a t u r a t e d (NH4)2SO 4 a t p H 7. Conditions are described in the text.
crystals and the speed of shaking. When the esterolitic activity of chymotrypsin crystals was measured in 80% saturated (NH4)2SO4 at pH 7, using acetyl-L-tyrosine ethyl ester as substrate, the catalytic activity was found to be 17-2o % of the enzyme activity in solution. Also it was found that amorphous chymotrypsin in the solid state possessed enzymic activity equal to 2o-25 % of that of soluble chymotrypsin using acetyl-~-tyrosine hydrazide as substrate. The rate of hydrolysis of acetyl-L-tyrosine hydrazide substrate by crystalline chymotrypsin was found to be directly proportional to the amount of solid enzyme present, as illustrated in Fig. I. A linear relationship was obtained in the range of 1-6 mg of solid crystalline enzyme present per sample. A zero order reaction of the hydrolysis of acetyl-L-tyrosine hydrazide was determined with varying amounts of chymotrypsin crystals. The rate of hydrolysis was linear for a period of slightly longer than 30 rain as shown in Fig. 2. These observations illustrate a similarity between the activity of chymotrypsin crystals and the activity of chymotrypsin in solution. Quite recently SIGLER AND Biochim. Biophys. Acta, 89 (1964) 364-366
366
SHORT COMMUNICATIONS 0.5
0.4
~'O.2 tO tO
~6
8m < 0.1
o
~b
~o
Time (min)
~o
~o
~o
do
Fig. 2. The initial velocity- of the hydrolysis of acetyl-L-tyrosine hydrazide by crystalline chymotrypsin in the solid phase in 80% saturated (NH4)~SO 4 at pH 7 obtained by plotting the absorbancy in function of time. Conditions are described in the text.
SKINNER5 found that crystals of chymotrypsin react stochiometrically with diisopropylfluorophosphate to give the same phosphorylated enzyme as that prepared in solution. DOSCHER AND RICHARDSe have recently reported catalytic activity in the crystalline state of another hydrolytic enzyme, ribonuclease (EC 2.7.7.16). Ribonuclease in the crystalline state in 9 ° % saturated (NH4),SO 4 was found to be catalytically active using uridine 2' :3' cyclic phosphate as substrate. The present study shows that chymotrypsin in the crystalline state is catalytically active suggesting that the crystalline enzyme is in an enzymatically active conformation. The author wishes to thank Miss D. RlZOK for her assistance with some of the early experiments, and Miss E. ARTHURfor her careful and expert technical assistance. The author is also grateful to Dr. P. SIGLER from the National Institutes of Health for the communication of his results prior to publication. This investigation has been supported in part by a research grant from the Medical Research Council of Canada (I96I-I963).
Research Laboratory, Department of Pathology, St. Mary's Memorial Hospital, Montreal (Canada)
J . KALLOS
1 F. ~¢~. I~ICHARDS, A ~ . Rev. Biochem., 32 (1963) 369. 2 j . KENDREW, Brookhaven Syrup. Biol., 15 ~I962) 216. 8 j . KENDREW, Science, 139 (1963) 1259. 4 G. W. SCI-IWERT AND TAKENAKAY, Biochim. Biophys. Acta, 16 (1955) 57 o. s p. ]3. SIGLER AND H. C. W. SKINNER, Biochem. Biophys. Res. Commun., in t h e press. s M. DOSCHER AND V. M. RICHARDS, J. Biol. Chem., 238 (1963) 2399.
Received April i3th , 1964 Biochim. Biophys. Acta, 89 (I964) 364-366
SHORT COMMUNICATIONS
In summary, the 50% ammonium sulfate precipitate from a homogenate of adrenal cortical tissue showed only 2oa-hydroxysteroid dehydrogenase activity. This study was supported by Grant AM-o3269, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U.S. Public Health Service.
Departments of Endocrinology and Biochemistry, Southwest Foundation for Research and Education, San Antonio, Texas (U.S.A.)
C. MATTHIJSSEN J. E. MANDEL
P. T. SEIDEN
x C. MATTHIJSSEN AND J. E. MANDEL, Biochim. Biophys. Acta, 82 (1964) 138. 2 L. R. AX~LROD, J. Biol. Chem., 2o 5 (1953) 173. 3 A. ZAFFARONI AND B. BURTON, J. Biol. Chem., 193 (1951) 749. 4 L. R. AXELROD, Anal. Chem., 27 (1955) 13o5. s W. G. WIEST ANn R. B. WILCOX, J. Biol. Chem., 236 (1961) 2425.
Received March 3Ist, 1964
Biochim. l~iophys. Acta, 89 (1964) 363-364
sc 63048
Catalytic activity of chymotryp$in in crystalline state Enzyme molecules generally function in dilute aqueous solution. However, it would be interesting to know the relationship between a protein molecule in solution and one in the crystalline state 1. It is now possible by means of X-ray diffraction to establish the complete three-dimensional structure of protein molecule in this latter state, as illustrated in the case of myoglobin 2,s. With this in view, a comparison of the enzymic activity of protein molecules in the crystalline state with those in solution was undertaken. Chymotrypsin (EC 3.4.4.5) in the crystalline state was found to be catalytically active with enzymic properties similar to those obtained in dilute solution .The enzymic activity of the crystals was measured by using acetyl-L-tyrosine hydrazide and acetyl-L-tyrosine ethyl ester as substrates * in 8o% saturated (NH4),SO ,. a-Chy{-notrypsin (Mann, salt-free, 3XCryst) was crystallized at room temperature from 0.5 s~turated (NH4),SO 4 yielding crystals approx, o.i-o.2 mm in width and 0.5-0.8 mm in length. A typical enzyme-assay was run as follows : crystals of chymotrypsin (I mg) were suspended in a solution (4 ml) of 2. IO-3 M acetyl-L-tyrosine hydrazide substance in 80% saturated (NH4)2SO , at pH 7. The reaction mixture was incubated at 25 ° with mild shaking. After a given time interval (30 min), crystals were allowed to settle for about a minute and aliquots of the supernatant solution (o.25 ml) were withdrawn and the hydrazine liberated was determined colorimetrically by the use of Ehrlich's Reagent. Results were compared with those obtained with a standard chymotrypsin solution to which 80% saturated (NH4),SO 4 had to be added. (The presence of the 80% saturated (NH4),SO , in the solution increased the rate of enzymic hydrolysis by 25-30%. ) The weight of the solid enzyme after the assay was determined spectrophotometricaUy* by measuring tile protein concentration of the dissolved crystals. A control experiment was performed to determine the solubility of
Biochim. Biophys. Aaa, 89 (1964) 364-366
365
SHORT COMMUNICATIONS
the enzyme crystals in the assay mixture by measuring the enzymic activity and protein concentration of the 80% saturated (NH4)~SO ~ supernatant solution of the crystals. The solubility of the crystals (approx. o.0o8 mg in solution for I mg of crystalline enzyme) was found to be less than 1%. This could thus only account for a small fraction of the observed activity. All results expressing enzymic activity of chymotrypsin in crystalline state were corrected accordingly. From this assay, the rate of hydrolysis of acetyl-e-tyrosine hydrazide by I mg of the crystals of chymotrypsin was equal to the activity of o.18 mg of chymotrypsin in solution or I 8 % activity as compared with the same amount of enzyme in solution. The activity was found to vary from I5-2I % depending on the different batches of 0.7 0.6 0.5
E•0.4
U3 tO ~- 0.3
~ o.2 0
~ o.1
<
0
o
I
4.0 1.o 2'.0 3'.0 ' Crystals of chymotvypsin (rag pep sample)
5t.0
I
6.0
Fig. I. R a t e of hydrolyses of acetyl-L-tyrosine hydrazide s u b s t r a t e as a function of the a m o u n t of crystalline c h y m o t r y p s i n p r e s e n t in the solid phase in 8 o % s a t u r a t e d (NH4)2SO 4 a t p H 7. Conditions are described in the text.
crystals and the speed of shaking. When the esterolitic activity of chymotrypsin crystals was measured in 80% saturated (NH4)2SO4 at pH 7, using acetyl-L-tyrosine ethyl ester as substrate, the catalytic activity was found to be 17-2o % of the enzyme activity in solution. Also it was found that amorphous chymotrypsin in the solid state possessed enzymic activity equal to 2o-25 % of that of soluble chymotrypsin using acetyl-~-tyrosine hydrazide as substrate. The rate of hydrolysis of acetyl-L-tyrosine hydrazide substrate by crystalline chymotrypsin was found to be directly proportional to the amount of solid enzyme present, as illustrated in Fig. I. A linear relationship was obtained in the range of 1-6 mg of solid crystalline enzyme present per sample. A zero order reaction of the hydrolysis of acetyl-L-tyrosine hydrazide was determined with varying amounts of chymotrypsin crystals. The rate of hydrolysis was linear for a period of slightly longer than 30 rain as shown in Fig. 2. These observations illustrate a similarity between the activity of chymotrypsin crystals and the activity of chymotrypsin in solution. Quite recently SIGLER AND Biochim. Biophys. Acta, 89 (1964) 364-366
366
SHORT COMMUNICATIONS 0.5
0.4
~'O.2 tO tO
~6
8m < 0.1
o
~b
~o
Time (min)
~o
~o
~o
do
Fig. 2. The initial velocity- of the hydrolysis of acetyl-L-tyrosine hydrazide by crystalline chymotrypsin in the solid phase in 80% saturated (NH4)~SO 4 at pH 7 obtained by plotting the absorbancy in function of time. Conditions are described in the text.
SKINNER5 found that crystals of chymotrypsin react stochiometrically with diisopropylfluorophosphate to give the same phosphorylated enzyme as that prepared in solution. DOSCHER AND RICHARDSe have recently reported catalytic activity in the crystalline state of another hydrolytic enzyme, ribonuclease (EC 2.7.7.16). Ribonuclease in the crystalline state in 9 ° % saturated (NH4),SO 4 was found to be catalytically active using uridine 2' :3' cyclic phosphate as substrate. The present study shows that chymotrypsin in the crystalline state is catalytically active suggesting that the crystalline enzyme is in an enzymatically active conformation. The author wishes to thank Miss D. RlZOK for her assistance with some of the early experiments, and Miss E. ARTHURfor her careful and expert technical assistance. The author is also grateful to Dr. P. SIGLER from the National Institutes of Health for the communication of his results prior to publication. This investigation has been supported in part by a research grant from the Medical Research Council of Canada (I96I-I963).
Research Laboratory, Department of Pathology, St. Mary's Memorial Hospital, Montreal (Canada)
J . KALLOS
1 F. ~¢~. I~ICHARDS, A ~ . Rev. Biochem., 32 (1963) 369. 2 j . KENDREW, Brookhaven Syrup. Biol., 15 ~I962) 216. 8 j . KENDREW, Science, 139 (1963) 1259. 4 G. W. SCI-IWERT AND TAKENAKAY, Biochim. Biophys. Acta, 16 (1955) 57 o. s p. ]3. SIGLER AND H. C. W. SKINNER, Biochem. Biophys. Res. Commun., in t h e press. s M. DOSCHER AND V. M. RICHARDS, J. Biol. Chem., 238 (1963) 2399.
Received April i3th , 1964 Biochim. Biophys. Acta, 89 (I964) 364-366